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)     

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.     

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.     

Photosynthetic properties of leaves of a yellow green mutant of wheat compared to its wild type

We investigated several photosynthetic parameters of a virescent mutant of durum wheat and of its wild-type. Electron transport rate to ferricyanide was the same in the two genotypes when expressed on leaf area basis while O₂ evolution of the leaf tissue in saturating light and CO₂ was slightly higher in the yellow genotype. RuBPCase was also slightly higher. Quantum yield per absorbed light was similar in the two genotypes. P700 and Cyt f were less concentrated in the mutant while PS II was only marginally lower. The light response curve of CO₂ assimilation indicated higher level of photosynthesis of the mutant in high light, which corresponded to a lower non-photochemical quenching compared to the wild-type. It is concluded that the reaction centres, cyt f and chlorophyll are not limiting factors of electron transport in wheat seedlings and that electron transport capacity is in excess with respect to that needed for driving photosynthesis. Since the differences in photosynthesis reflect differences in RuBPCase activity, it is suggested that this enzyme limits photosynthesis in wheat seedlings also at high light intensities.Abbreviations cyt f cytochrome f - chl chlorophyll - PS II photosystem II - Pn_max maximum photosynthesis - RuBCase Ribulose, 1-5,bisphosphate carboxylase     

Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth

Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the reversible binding of virginiamycin M to ribosome and particle functions after removal of the antibiotic

Type A synergimycins (VM) were shown to act catalytically and to induce two ribosomal alterations: (a) inability to promote polypeptide synthesis; (b) high-affinity binding of type B synergimycins (VS). A claim for irreversible binding of type A synergimycins to ribosomes has promoted the present reinvestigation. Submission of ribosomes from VM-treated bacteria to a purification procedure (supposed to remove the drug, according to a low association constant previously reported) yielded particles still holding residual VM. The formation of VM.ribosome complexes, more stable than previously inferred but without covalent linkage, was deduced from the extractability of complexed VM by organic solvents. Moreover, incubation of these complexes with increasing amounts of anti-VM immunoglobulins progressively restored ribosome activity in protein synthesis. Binding of VS to ribosomes, by fluorimetric titrations in the presence of substoichiometric concentrations of VM, was incompatible with catalytic action of type A synergimycins. Ribosomes from VM-treated bacteria displayed also a higher affinity for VS than did control ribosomes. This property did not disappear when ribosome.VM complexes were incubated with anti-VM IgG, nor when VM-IgG complexes were withdrawn from the reaction mixture by protein A-agarose binding. We can conclude that VM binding produces: (1) an inhibition of ribosome-promoted peptide bond formation, which occurs only in the presence of the drug; and (2) an increase of ribosome affinity for VS, which lasts after VM removal. The linkage of this drug with ribosomes is tight but reversible and its action is stoichiometric.     

Mono and two-dimensional 500-MHz characterization of synthetic bombesin and related peptides in DMSO and DMSO-water

The proton nmr characterization of bombesin (BBS) and of two peptide fragments corresponding to the (1-6) and (6-14) sequences has been carried out at 500 MHz in dimethyl sulfoxide (DMSO-d6) using two-dimensional (2D) homo and 1H-13C heterocorrelated techniques. All resonances in the nmr spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants are quite similar and around 7.8-8.2 Hz, pointing to an unfolded structure in DMSO-d6. The possibility of secondary structures in highly viscous mixtures of DMSO-d6-water was investigated. The existence of sequential nuclear Overhauser enhancement (NOE) effects in the C-terminal nonapeptide section may indicate a preferential site for secondary structuring.     

Genomic distribution of copia-like transposable elements in somatic tissues and during development of Drosophila melanogaster

The genomic distribution of elements of the copia, 412, B 104, mdg 1, mdg 4 and 1731 transposon families was compared by the Southern technique in DNA preparations extracted from brains, salivary glands and adult flies of two related Drosophila lines. The copia, 412 and mdg 1 sequences were also probed in DNA from sperm, embryos, and 1st and 2nd instar larvae. The homogeneity of the patterns observed shows that somatic transposition is unlikely to occur frequently. A correlation between mobility and the euchromatic or heterochromatic location of transposable elements is discussed. In addition, an explanation of the variable band intensities of transposable elements in Southern autoradiographs is proposed.     

N-terminal region of Proteus mirabilis glutathione transferase is not homologous to mammalian and plant glutathione transferases

The N-terminal amino acid sequence of glutathione transferase, Pm-GST-6.0, purified from Proteus mirabilis [(1988) Biochem. J. 255, 971-975] up to residue 38 and a comparative peptide fingerprint are reported. No obvious homology with the sequences of alpha, pi and mu classes of mammalian glutathione transferases as well as with those of plant glutathione transferases has been noted. These results suggest that the classification so far adopted for glutathione transferases cannot be extended to the bacterial enzyme.