Incorporation of Radioactive Seleno-(75Se)-Methionine into Mumps Virus

Mumps virus was grown in embryonated chicken eggs in the presence of radioactive seleno-(⁷⁵Se)-methionine. Virus in the allantoic and amniotic fluids was concentrated in a sucrose density gradient, and a peak of viral material coincided with a significant peak of ⁷⁵Se-radioactivity. The radioactivity was acid-insoluble and remained associated with the virus after purification by erythrocyte adsorption and elution and centrifugation on a second sucrose density gradient. After amino-acid hydrolysis of the radioactive virus, only ⁷⁵Se-methionine was recovered by chromatographic analysis. These results demonstrate that the radioactive ⁷⁵Se-methionine was incorporated into protein of infectious mumps virus.     

Synthesis of [75Se]trimethylselenonium iodide from [75Se]selenocystine

N-Acetylglucosamine-6-sulfate sulfatase activity was assayed by incubation of the radiolabeled monosaccharide N-acetylglucosamine [1-14C]6-sulfate (GlcNAc6S) with homogenates of leukocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with N-acetylglucosamine-6-sulfate sulfatase deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses. The assay clearly distinguished affected homozygotes from normal controls and other mucopolysaccharidosis types. The level of enzymatic activity toward GlcNAc6S was compared with that toward a sulfated disaccharide and a sulfated trisaccharide prepared from heparin. The disaccharide was desulfated at the same rate as the monosaccharide and the trisaccharide at 30 times that of the monosaccharide. Sulfatase activity toward glucose 6-sulfate and N-acetylmannosamine 6-sulfate was not detected. Sulfatase activity in fibroblast homogenates with GlcNAc6S exhibited a pH optimum at pH 6.5, an apparent Km of 330 mumol/liter, and inhibition by both sulfate and phosphate ions. The use of radiolabeled GlcNAc6S substrate for the assay of N-acetylglucosamine-6-sulfate sulfatase in leukocytes and skin fibroblasts for the routine enzymatic detection of the Sanfilippo D syndrome is recommended.     

[75Se]selenite and [75Se]selenate fluctuations during the development of murine hepatoma

The distribution of selenate and selenite selenium in C57L/J mice during the progression of BW7756 murine hepatoma was investigated using intra-ocular injection with the oxyanions labeled with ⁷⁵Se radioisotope. Comparison is made with the normal distribution of selenium studied by the RIXRF method. The trace elemental profiles, TEP, for the two oxidation states are compared in healthy and disease states. It has been found that the presence of tumor significantly changes the level of tracer in various uninvolved organs. These changes are prominent in the early growth phase of the tumor. The two oxidation states show differences in the TEP for kidney, spleen, stomach and testes.     

Assessment of platelet production with 75Se selenomethionine

Incorporation of intravenously injected ⁷⁵Se selenomethionine into platelets has been found to vary with alterations in the rate of platelet production. It appears to label newly produced platelets during their formation in megakaryocytes and provides a method by which thrombopoiesis may be studied in vivo. This technique may be applicable to clinical studies of disordered platelet production.     

Effect of lead on the intestinal absorption of sodium selenite and selenomethionine (75Se) in chicks

The present study was designed to investigate the interactions of lead with the intestinal absorption of selenium in chicks. The absorption of⁷⁵Se-selenite and⁷⁵Se-methionine was determined in 3-wk-old white Leghorn cockerels using both thein situ ligated intestinal loop procedure and oral administration of the isotopes. The highest lead concentration (1 mM) significantly reduced the percentage of⁷⁵Se-selenite absorbed from thein situ ligated duodenal loop, but did not influence the retention of the orally administered isotope. Neither was the absorption of⁷⁵Se-methionine affected by the presence of Pb in the intraduodenal dosing solution. The above experiments suggest that the direct luminal interaction of lead with the absorption of selenium is not of practical importance. On the other hand, feeding 1000 ppm Pb in the diet prior to the measurement of selenium absorption significantly reduced the transfer of the radioactive selenite from the intestinal lumen to the body. The mechanism of this effect involved enhanced retention of the⁷⁵Se-selenite by the intestinal tissue. The inhibitory effect of lead exposure on selenium absorption also appeared to be alleviated by the increase in the dietary selenium content.     

Uptake of75Se in cataractous rat lens proteins

The purpose of the present study was to measure the pattern of uptake of⁷⁵Se into proteins in normal rat lenses and into the proteins of lenses with selenite-induced cataract. Ten-day-old suckling rats received a single injection of⁷⁵Se with or without a cataractous dose of cold carrier sodium selenite. Four days after injection, the proteins from excised lenses were counted for⁷⁵Se radioactivity and subjected to gel permeation chromatography, amino acid analyses, and mass spectrometry. All three soluble crystallin lens proteins took up⁷⁵Se in both normal and cataractous lenses. However, cataractous lenses did not take up⁷⁵Se into a soluble protein in which major quantities of⁷⁵Se were taken up in normal rats. Futhermore,⁷⁵Se in the gamma-crystallins was associated with an unusual acidic amino acid. It was concluded that selenium metabolism by lens proteins may be unusual compared to other soft tissues.     

Study of mammalian selenocysteyl-tRNA synthesis with [75Se]HSe

The mechanisms of the synthesis of mammalian selenocysteyl-(Scy)-tRNA were studied using [75SE]H2Se. H2Se was prepared from [75Se]selenite, glutathione, NADPH and glutathione reductase, and was purified by chromatography. It was confirmed that this H2Se was a Se donor in the reaction of the synthesis of Scy-tRNA. [75Se]Scy, liberated from aminoacyl-tRNA, was analyzed by TLC on silica gel an subsequent autoradiography. The activity of Scy-tRNA synthesis was found in the supernatant at 105,000 x g of the murine liver extract, but not in the precipitate. The supernatant was chromatographed on DEAE-cellulose, and the activity was eluted at a concentration of 0.17 M KCl. This position is at the front shoulder of the peak of seryl-tRNA synthetase which was eluted at 0.20 M KCl. Major serine tRNA(IGA) is not a substrate on which to synthesize Scy-tRNA, but natural opal suppressor serine tRNA is. On a chromatographic pattern of a Scy-tRNA preparation on Sephacryl S-200, the radioactivity of 75Se was eluted at the tRNA peak. This showed that Scy bound to tRNA. The active protein fraction from DEAE-cellulose did not contain tRNA kinase, therefore Scy-tRNA must be directly synthesized from seryl-tRNA, not through phosphoseryl-tRNA. This mechanism is similar to that seen in Escherichia coli [1991, J. Biol. Chem. 266, 6324].     

Se-(8-azidoadenosyl)[75Se]selenomethionine as a photoaffinity label for S-adenosylmethionine binding proteins.

A method is described for the synthesis and purification of the photoaffinity label Se-(8-azidoadenosyl)[75Se]selenomethionine. This photoaffinity label can be used to specifically and covalently label the S-adenosylmethionine binding site of proteins that use this cofactor, as exemplified by labeling of thioether methyltransferase. By utilizing the gamma-emitting isotope of selenium, Se-(8-azidoadenosyl)[75Se]selenomethionine eliminates the need for the impregnation of acrylamide gels with fluorographic enhancers and dilution of liquid samples into scintillation cocktails, as is required with the commonly used methyl-3H-labeled and 35S-labeled S-(8-azidoadenosyl)methionine.     

CD 271 (P75 neurotrophin receptor)

P75NTR (or CD271) is a member of the Tumor Necrosis Factor receptor (TNFR) super family of transmembrane proteins that share significant homology in their extracellular domains. Subsets of TNF receptors, including CD271, have a cytoplasmic death domain, although CD271 has unique intracellular structure and downstream signaling partners. CD271 is also differentiated from other members of the TNFR receptor family in that it binds pro and mature neurotrophins and affects the growth, differentiation and death of the nervous system. The ligands for CD271 are neurotrophins, which are Nerve Growth Factor (NGF), Brain-Derived Growth factor (BDNF), Neurotrophin 3 (NT3) and Neurotrophin 4/5 (NT4/5). Recent studies have provided evidence that CD271 also serves as a receptor for the pro-forms of these neurotrophins.     

A comparison of the uptake of [75Se]selenite, [75Se]selenomethionine and [35S]methionine by tissues of ewes and lambs.

The fate of selenium, given as Na2(75)SeO3, or [75Se]selenomethionine, and of [35S]methionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal. Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis. Small, though significant amounts of selenium, derived from Na2(75)SeO3, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs. The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.     

p75(NGFR) and TrkA receptors collaborate to rapidly activate a p75(NGFR)-associated protein kinase.

The role of the low affinity nerve growth factor receptor (p75(NGFR)) in NGF-mediated signaling is not yet understood. Here we show by co-immunoprecipitation that NGF activates a protein kinase that is directly associated with p75(NGFR) in dorsal root ganglion (DRG) cells and PC12 cells in culture. Two proteins of 120 and 104 kDa constitute the majority of this activity. In PC12 cells, TrkA activation was necessary to elicit p75(NGFR)-associated kinase activity. Although NGF binding to p75(NGFR) was not necessary for kinase activation, it accelerated the activation of the kinase at low NGF concentrations. Deletion analysis showed that a 43 amino acid region in the cytoplasmic domain of p75(NGFR) was responsible for this effect. These findings show that p75(NGFR) accelerates TrkA-mediated signaling and, in addition, demonstrate that p75NGFR and TrkA collaborate to activate a previously undescribed p75(NGFR)-associated protein kinase.     

Yakov Abramovich Altman (To the 75-Anniversary)

Jubilee Dates

Yakov Abramovich Altman (To the 75-Anniversary)