Studies with insulin and insulin-like growth factor-I show that the increased labeling of phosphatidylinositol-3,4-bisphosphate is not sufficient to elicit the diverse actions of epidermal growth factor on MA-10 Leydig tumor cells

In a recent publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells rapidly leads to an increase in the incorporation of [3H]inositol-derived radioactivity into an unusual lipid that was identified as phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2). Other ligands that are known to bind to MA-10 cells, such as hCG and arginine vasopressin, however, did not elicit this effect. Inasmuch as mEGF modulates the differentiated functions of MA-10 cells in a number of ways, our findings raised the possibility that PI-3,4-P2 may be an intracellular mediator of these actions of mEGF. In an attempt to answer this question, we set out to determine if other ligands increase the labeling of PI-3,4-P2 in MA-10 cells prelabeled with [3H]inositol, and if such ligands mimic the diverse biological actions of mEGF on these cells. The experiments presented herein show that insulin, insulin-like growth factor-I, and transforming growth factor-alpha increase the labeling of PI-3,4-P2 in MA-10 cells, but only transforming growth factor-alpha mimics the actions of mEGF on the differentiated functions of MA-10 cells. We conclude that an increase in the labeling of PI-3,4-P2 is not sufficient to elicit these actions of mEGF.     

Distribution of Thiamine, Thiamine Phosphates, and Thiamine Metabolizing Enzymes in Neuronal and Glial Cell Enriched Fractions of Rat Brain

The distribution of thiamine, thiamine phosphoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydrolyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions.     

Bovine pancreatic trypsin inhibitor and homologous polypeptide inhibitors in nephron cells.

Bovine pancreatic trypsin inhibitor (BPTI, aprotinin) is a fifty-eight amino acid polypeptide, which is present together with related molecular isoforms in various bovine organs. In the present study these protease inhibitors were isolated from bovine kidney by affinity chromatography on immobilized trypsin and a subsequent FPLC step. Due to their electrophoretic, structural, and inhibitory properties, the inhibitors were strictly similar to the polypeptides identified previously in other bovine organs. Immunohistochemical experiments showed a widespread localization of these polypeptides in nephron epithelial cells (proximal and distal tubules, loop of Henle, collecting tubules).     

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mini-myoglobin: preparation and reaction with oxygen and carbon monoxide

A domain of 108 amino acid residues (32 to 139), obtained by digestion of horse heart apomyoglobin with clostripain, was found to bind protoheme in a 1 to 1 molar ratio. This domain is 33 amino acid residues larger than the protein segment encoded by the central exon in seal myoglobin. Flash photolysis experiments have shown that reconstituted "mini-myoglobin" is very similar to myoglobin in the combination reaction with carbon monoxide and with oxygen, and in the oxygen replacement reaction by carbon monoxide. These experiments provide for the first time direct evidence for the presence of a structural and functional domain, closely corresponding to the segment encoded by the central exon of the myoglobin gene, which contains the information for binding the natural heme and for maintaining the native folding typical of a respiratory protein.     

Tubular structures associated with the endothelial endoplasmic reticulum in glomerular capillaries of Rhesus monkey and nephritic man

Summary Round bodies, tubular profiles and crystalline images have been studied by electron microscopy in the endothelium of seven normal young Rhesus monkeys and in the renal glomerular endothelium of two nephritic human patients. The crystalline images are most frequently formed by aggregation of round bodies, 200–240 Å in diameter. In Rhesus monkeys a variety of crystalline images are seen. On the contrary, in nephritic patients round bodies and tubular profiles are rare and less organized. In the glomerular endothelium of two normal men they were not found.The results obtained suggest that the round bodies, the tubular profiles and the crystalline images result from sectioning of a system of undulating tubules associated with the smooth endoplasmic reticulum. In nephritic patients the formation of such a tubular system could represent a change taking place within the affected cells as a pathologic response to the disease.This work was supported by U.S. Public Health Service (N.H.I.), Grant AI-04527-03-04, and by the Consiglio Nazionale delle Ricerche (C.N.R.), Contributo 115/0815/0-1365. The Authors are greatly indebted to Miss Hermina Spiele for skilful technical assistance.     

A re-evaluation of the mesangial cells of the renal glomerulus

Summary The topographical localization of the mesangial cells in renal glomeruli of rats, and their relationships with the structures at the hilus of the glomerulus were studied in this investigation. It was observed that the mesangial cells occupy a parietal position in the wall of the glomerular capillaries, and that they are anatomically continuous with the smooth muscle cells of the tunica media of the glomerular arterioles.This study was supported by a United States Public Health Service Grant AM 08628 (Institute of Arthritis and Metabolic Diseases).     

Stomatal and cuticular transpiration of beans (Phaseolus vulgaris M.) attacked by rust (Uromyces appendiculatus [Pers.] Link)

The changes of stomatal and cuticular transpiration of bean plants were investigated by graphical transpiration curves method (Slavík 1958). Bean leaves were infected by fungusUromyces appendiculatus (Pers.) Link. After the infection the intensity of stomatal transpiration had a decreasing tendency. Beginning with the sixth day after infection, the proportion of stomatal and cuticular transpirations becomes more expressive, i.e. the leaves transpire more by cuticles than by stomata. The higher share of cuticular transpiration brings extensive water relations to the diseased plants.     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.