Increased efflux rather than oxidation is the mechanism of glutathione depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Incubation of isolated hepatocytes in the presence of either the parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its putative toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) led to a depletion of intracellular reduced glutathione (GSH), which was mostly recovered as glutathione disulfide (GSSG). However, both MPTP- and MPP+-induced glutathione perturbances were relatively unaffected by the prior inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), suggesting that intracellular oxidation was not the major mechanism involved in the GSH loss. Inclusion of cystine in the incubation mixtures revealed a time-dependent formation of cysteinyl glutathione (CySSG), indicating that an increased efflux was mostly responsible for the MPTP- and MPP+-induced GSH depletion. Therefore, the measurement of GSSG, which is apparently formed extracellularly, was not associated with oxidative stress.     

Involvement of erythrocyte skeletal proteins in the modulation of membrane fluidity by phenothiazines

The effects of phenothiazines (chlorpromazine, chlorpromazine sulfoxide, and trifluoperazine) and antimitotic drugs (colchicine and vinblastine) on the erythrocyte membrane have been investigated. Chlorpromazine and trifluoperazine induced a dose-dependent increase in the freedom of motion of stearic acid spin-labels bound to both intact erythrocytes and ghosts, but did not affect the freedom of motion of stearic acids bound to vesicles depleted of spectrin and actin or of ghosts resealed with anti-spectrin antibodies. Further, chlorpromazine and trifluoperazine were able to eliminate a protein 4.1 dependent membrane thermal transition detected by stearic acid spin-labels at 8.5 +/- 1.5 degrees C. Antimitotic drugs and chlorpromazine sulfoxide did not change either the freedom of motion of stearic acid spin-labels or the 8.5 degrees C membrane thermal transition. Results indicate the involvement of skeletal proteins as possible membrane target sites of biologically active phenothiazines and suggest that the control of stearic acid spin-label freedom of motion is mediated by the spectrin-actin network and the proteins that link the skeletal network to the membrane.     

Properties of carboxymethylated cross-linked hemoglobin A

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractility, ATP, and creatine phosphate during myocardial ischaemia and reperfusion: the effects of adenosine and inhibition of adenosine catabolism in the dog heart

Provision of AMP or adenosine to heart cells during recovery from episodes of myocardial ischaemia accelerates physiological, biochemical, and structural recovery. Inhibition of adenosine loss from the tissue during ischaemia should have a similar effect. This hypothesis was tested in dog heart by infusion of adenosine and inhibitors of adenosine catabolism prior to, during, and following ischaemia. Post-ischaemic recovery of ATP and contractile function was accelerated significantly by adenosine and by inhibitors of adenosine catabolism both singly and in combination. Contractility and ATP levels during ischaemia were also increased by these inhibitors.     

Successful pregnancy in a woman with congenital factor XIII deficiency treated with substitutive therapy. Report of a second case

A syndrome of marked fetal wastage is associated with congenital factor XIII deficiency in adult women. A previously unreported case of a woman with factor XIII deficiency is described, in which substitutive treatment with normal plasma or placental factor XIII concentrate permitted two normal pregnancies. Factor XIII activity was maintained above 1-2% with intermittent infusion of 300 ml to 450 ml of plasma every 14 days or of 500 units of concentrate every 21 days. This case confirms the only other case so far reported in which factor XIII substitutive therapy was able to permit a normal pregnancy in a woman with factor XIII deficiency and seems to suggest factor XIII to be involved in the process of annidation.     

Mesodermal metamerism in the teleost, Oryzias latipes (the medaka)

Previous studies of the metameric pattern in mesodermal tissues of chick, mouse, turtle, and amphibian embryos have indicated that segmental characteristics exist along the entire length of the embryo. This paper describes this phenomenon in a fish embryo, for some differences in the cranial segmental plan exist between the anamniote and the amniote embryos hitherto studied. Embryos of the cyprinodont, Oryzias latipes, were fixed at various times, the examined by means of stereo scanning electron microscopy. As in other vertebrate embryos, the first indication of mesodermal metamerism in this fish embryo is the occurrence of somitomeres, which are orderly, tandemly arranged units of uncondensed mesenchymal cells in the paraxial mesoderm. As many as ten somitomeres can be observed caudal to the last formed somite to the elongating tail region. In addition, 7 somitomeres are present rostral to the first definitive somite, which is segment number eight. As in other vertebrate embryos examined, somitomeres in Oryzias embryos are circular, bilaminar arrays of paraxial mesoderm that form before any indications of segmentation can be seen with the light microscope. In the trunk region these mesodermal units condense to give rise to definitive somites, but in the head they eventually disperse. Despite a fundamentally different mode of gastrulation and a relatively small number of cells in the newly formed somitomeres, cranial segmentation in Oryzias embryos was found to be more similar in number to the metameric pattern of the embryos of the bird, reptile, and mammal than to the situation found in the two amphibians studied thus far.     

Analysis of uric acid and oxypurines in normal subjects and in gout patients

The behavior of plasma and urine oxypurines (hypoxanthine and xanthine) and of uric acid has been studied in normal subjects and in gout patients. Oxypurines and uric acid were increased in the plasma of gout patients but only the urinary excretion of hypoxanthine was higher in this group. The interpretation of the observed variations is discussed.     

Purine metabolism: determination of PRPP-amidotransferase and PRPP-synthetase in lymphocytes from patients with chronic lymphatic leukemia (CLL)

Phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase is the "key anabolic enzyme" of purine nucleotide synthesis; PRPP synthetase connects the pentose cycle with the same pathway. We have studied their behavior in 5 control subjects and in 8 affected by CLL. Determination of PRPP amidotransferase was carried out through the evaluation of 14C-glutamic acid (released by 14C-glutamine) in the incubation mixture. PRPP synthetase was followed by adding ATP and ribose 5-phosphate to the incubation mixtures, and by evaluating the PRPP formed through the release of CO2 in a coupled reaction. In the case of PRPP-amidotransferase, our values are in the range reported in the literature: in patients affected by CLL, the enzyme activity is much higher and the increase is more evident when values referred to the patients, than when to the cells. Our values of PRPP synthetase are consistent with those of Peters and Veerkamp, but no definite conclusion is possible in the case of leukemic patients.     

The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.     

A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminal extracellular domains of human CD4 antigen, a cell surface glycoprotein, is reported. This region of CD4, particularly the first domain, has been identified as containing the binding region for the envelope gp120 protein of the human immunodeficiency virus. The model was predicted based on the sequence homology of each domain with the variable light chain of immunoglobulins. The framework beta-sheet regions were taken from the crystal coordinates of REI. For one region in the first domain of CD4 there was an ambiguity in the alignment with REI and two alternate models are presented. Loops connecting the framework were modelled from fragments selected from a database of main chain coordinates from all known protein structures. Residues identified as involved in binding gp120 have been located in several other studies within the first domain of CD4. Epitopes from eight monoclonal antibodies have been mapped onto residues in both domains. Competition of these antibodies with each other and with gp120 can be interpreted from the structural model.