Gas chromatographic measurement of urinary 5-fluoro-2'-deoxyuridine levels after barium salt precipitation and sephadex LH-20 cleanup

A fluorescent photoaffinity label—8-azido-1-N⁶-etheno-adenosine 5′-triphosphate (8-N₃ε ATP)—for ATP-binding proteins has been synthesized. The effectiveness of the label is demonstrated with F₁ATPase from Micrococcus luteus. 8-N₃ε ATP is a substrate for the enzyme in the presence of bivalent cations. Ultraviolet irradiation of F₁ATPase in the presence of the label and Mg²⁺ ions inhibits the enzyme irreversibly. The fluorescent label is bound preferentially to the β subunit of the enzyme. Labeling and inactivation are decreased by protection with ATP or ADP.     

Branchial Cl transport, anion-stimulated ATPase and acid-base balance inAnguilla anguilla adapted to freshwater: Effects of hyperoxia

Summary Freshwater eel gills are notorious for their limited ability to pump chloride. As a result there is a considerable discrepancy between the Na⁺ and Cl^– plasma levels, and plasma HCO₃ ^– and blood pH are relatively high in this species.When eels are kept in tanks aerated with pure oxygen, significant alterations in blood acid-base balance, an increase in plasma pCO₂ and a decrease in blood pH, are observed. In fish studied after 3 weeks hyperoxia, the decrease in blood pH is compensated by an increase in plasma HCO₃ ^–. Such fish exhibit a Cl^– influx 5 times higher than that observed in normoxic fish. This Cl^– influx is readily inhibited by addition of SCN^– to the external medium.An anion-stimulated ATPase activated by HCO₃ ^– and by Cl^– and inhibited by SCN^– was recently described in membrane fractions of the gills ofCarassius auratus, a fish noted for its high Cl^– pumping rate. This enzyme is also found in the gills of the eel. While the maximal rates of enzyme activation by HCO₃ ^– and by Cl^– are similar inCarassius andAnguilla, the affinity of the enzyme for Cl^– is 25 times higher inCarassius. In the microsomal fraction of the hyperoxic eel gills, the maximal anionstimulated ATPase activity remains unchanged but HCO₃ ^– affinity decreases by 50%, while Cl^– affinity increases 5 times. Thus some characteristics of this ATPase seem to be closely related to the Cl^– pump activity exhibited by the gill in fresh water.     

1H NMR studies at 360 Mhz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI)

In the ¹H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformations are discussed.     

Inhibition of haem synthesis caused by cobalt in rat liver. Evidence for two different sites of action.

Cobalt inhibits liver haem synthesis in vivo by acting at least two different sites in the biosynthetic pathway: (1) synthesis of 5-aminolaevulinate and (2) conversion of 5-amino-laevulinate into haem. The first effect is largely, if not entirely, due to inhibition of the activity of 5-aminolaevulinate synthase, rather than to inhibition of the formation of the enzyme. The second effect results from diversion of 5-aminolaevulinate into an unidentified liver pool with solubility properties similar to those of cobalt protoporphyrin.     

Loss of haem from cytochrome P-450 caused by lipid peroxidation and 2-allyl-2-isoprophylacetamide. An abnormal pathway not involving production of carbon monoxide.

1. Microsomal preparations undergoing lipid peroxidation produce CO and lose haem from cytochrome P-450. 2. The amount of CO produced does not correlate with the amount of haem lost and, after pre-labelling of microsomal haem in its bridges with 5-amino[5-14C]laevulinate, the radioactivity lost from haem is not recorved as CO. 3. Similarly, when pre-labelled microsomal haem is destroyed by the action of 2-allyl-2-isopropylacetamide, no radioactivity is recovered as CO. In clear contrast, on degradation of haem by the haem oxygenase system, CO is produced in an amount equimolar to the haem lost. 4. It is concluded that (a) the CO produced during lipid peroxidation originates from a source different from haem and (b) the degradations of haem caused by lipid peroxidation and 2-allyl-2-isopropylacetamide do not involve to any significant extent evolution of the methene-bridge carbon of haem as CO.     

Dedifferentiation of lens epithelial cells in tissue culture

Lens epithelial cells can be kept in their original differentiated state or brought to dedifferentiation depending on the culture conditions. The different stages of differentiation can be identified using specific markers, namely the activity of steroid metabolizing enzymes, and the synthesis of specific structural lens polypeptides. For this reason lens epithelial cells in tissue culture provide a unique system for the study of the regulation of RNA and protein biosynthesis.Abbreviations dehydroepiandrosterone (DHEA)= 3-hydroxy-5-androsten-17-one - androstenediol (ADIOL)= 5-androstene-3, 17-diol - androstenedione(ADION)= 4-androstene-3, 17-dione     

Sertoli cells of adult rats “in vitro”

Summary A cell line obtained from isolated seminiferous tubules of adult rat testis has been studied in vitro over a period of 35 days.Light and electron microscopic studies performed from hour 2 to the end of culture have shown the presence of a monomorphic cell population. After 5–6 days of culture the cells formed a monolayer. The cytoplasm of the cells contained numerous lipid bodies and produced numerous projections. The nucleus showed several indentations and one or more nucleoli. From the 9th to the 15th day of culture the cells developed a large amount of endoplasmic reticulum, Golgi apparatus and aggregates of electron dense granules. From the 20th to 40th day the cell cultures progressively degenerated.Immunochemical analysis of the culture medium revealed the presence of estradiol-17, which reached its maximum production rate from the 8th day to the 18th day of culture. Corresponding to cell involution estradiol concentration underwent a rapid decrease.On the basis of morphological and biochemical data the cells could be considered Sertoli cells.This work was supported by Grants n. 74.00155.04 and n. 75.01224.04 from the Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and by Istituto di Ricerca F. Angelini, Rome, ItalyPart of this work was presented at the 10th Italian Congress of Electron Microscopy. Ostuni 1–4 October 1975The excellent technical assistance of Miss Laura Vassallo, Daniela Venturini and Mr. Massimo Rosati and Mario Termine is deeply appreciated     

Genetic studies of the Macushi and Wapishana Indians

Summary Blood samples from 509 Macushi and 623 Wapishana Amerindians of Northern Brazil and Southern Guyana have been analyzed with reference to the occurrence of rare variants and genetic polymorphisms of the following 25 systems: (i) Erythrocyte enzymes: acid phosphatase-1, adenosine deaminase, adenylate kinase-k, carbonic anhydrase-1, carbonic anhydrase-2, esterase A_1,2,3, esterase D, galactose-1-phosphate uridyltransferase, isocitrate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, nucleoside phosphorylase, peptidase A, peptidase B, phosphoglucomutase 1, phosphoglucomutase 2, phosphogluconate dehydrogenase, phosphohexoseisomerase, triosephosphate isomerase and (ii) Serum proteins: albumin, ceruloplasmin, haptoglobin, hemoglobin A, hemoglobin A₂ and transferrin. Fifteen different rare variants were detected, involving 11 of these systems. In addition, a previously undescribed variant of ESA_1,2,3 which achieves polymorphic proportions in both these tribes is described. Excluding this variant, the frequency of rare variants is 1.1/1000 in 12510 determinations in the Macushi and 4.7/1000 in 15 396 determinations in the Wapishana. The ESA_1,2,3, polymorphism was not observed in 382 Makiritare, 232 Yanomama, 146 Piaroa, 404 Cayapo, 190 Kraho and 112 Moro. Irregularities in the intratribal distribution of this polymorphism in the Macushi and Wapishana render a decision as to the tribe of origin impossible at present. Gene frequencies are also given for previosly described polymorphisms of 5 systems: haptoglobin, phosphoglucomutase 1, erythrocyte acid phosphatase, esterase D, and galactose-1-phosphate-uridyl-transferase.Research supported by the National Science Foundation and the Energy Research and Development Administration.     

Inhibition of compensatory kidney hypertrophy by hypothyroidism in the rat. Measurement of the mean diameter of glomeruli and proximal tubules]

The body, heart and kidney weights are reduced in the hypothyroid rat. In this animal, the diameter of the proximal tubule is significantly smaller than in euthyroid controls. The glomerular diameter is not affected by hypothyroidism. 21 days after uninephrectomy, the weight of the remaining kidney and the diameter of the proximal tubule increase significantly both in the hypothyroid and the euthyroid rats. But this compensatory renal hyperthrophy is definitely impaired in the hypothyroid animals.