Reactivity of an extremenly sterically crowded monofunctional Pt complex, [Pt(1-MeC-N3)3(H2O)]2+ (1-MeC=1-methylcytosine), toward model nucleobases and selectivity toward guanine in single- and double-stranded deoxyoligonucleotides

 Reactions of [Pt(1-MeC-N3)₃Cl]NO₃ (1-MeC-N3=1-methylcytosine, bound to Pt via N3) and the respective aqua species [Pt(1-MeC-N3)₃(H₂O)]²⁺ with the model nucleobases 9-ethylguanine (9-EtGH), 9-methyladenine (9-MeA), single-stranded 5′d(T₃GT₃), and double-stranded [5′d(GAGA₂GCT₂CTC)]₂ have been studied in solution by means of ¹H NMR spectroscopy, HPLC, and electrospray ionization mass spectrometry. Reactions are generally slow, in particular with the chloro species, and guanine is the only reactive base in the oligonucleotides. However, unlike (dien)Pt^II, which binds randomly to the guanines in the ds dodecamer, (1-MeC-N3)₃Pt^II binds selectively to the terminal guanine only, probably because base fraying takes place at the duplex ends. The X-ray crystal structures of [Pt(1-MeC-N3)₃(9-EtG-N7)]ClO₄·8H₂O (1b) and of [Pt(1-MeC-N3)₃(9-MeA-N7)](ClO₄)₂·0.5H₂O as well as NMR spectroscopic studies of [Pt(1-MeC-N3)₃(9-EtGH-N7)] (NO₃)₂·H₂O (1a) are reported. The tetrakis(nucleobase) complexes adopt a head-tail-head orientation of the three 1-MeC bases and an orientation of the fourth base (purine) that permits a maximum of intracomplex H bonds between exocyclic groups. As far as the guanine adduct (1a, 1b) is concerned, relative orientations of the four bases are identical in the model and in the oligonucleotide adduct. Received: 19 June 1998 / Accepted: 1 October 1998     

Redox sensitive cysteine residues in calbindin D28k are structurally and functionally important

Human calbindin D(28k) is a Ca(2+) binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cysteine-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D(28k) appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D(28k) binds Ca(2+) with lower affinity than does the reduced protein.     

Leptotrichia buccalis bacteremia in two patients with acute myelogenous leukemia

Leptotrichia buccalis is rarely implicated in systemic disease. We report two patients with clinically significant L. buccalis bacteremia which developed during the neutropenia secondary to chemotherapy. Based upon our experience, L. buccalis bacteremia should be considered in certain high-risk immunocompromised patients with mucositis and/or gingivitis.     

Caffeine-inhibitable control of the radiation-induced G2 arrest in L5178Y-S cells deficient in non-homologous end-joining

The two L5178Y (LY) sublines bear a heterozygous Tp53 mutation that affects its transactivation function. LY-S (radiation-sensitive) cells are deficient in double strand break (DSB) repair by non-homologous end-joining (NHEJ) and do not express p21^WAF1 (Cdkna1) either constitutively or after x-irradiation, in contrast to their radiation-resistant counterpart LY-R cells, which express p21^WAF1 constitutively. Radiation-induced G2 arrest in LY-S cells is very long (11 h/Gy) but 2 mM caffeine treatment shortens it, decreases the fraction of G2 cells and increases the fraction of apoptotic cells. The treatment also increases the DNA damage that is estimated with the comet assay 18 h after irradiation with 5 Gy (ca. 23% of the initial value for x-rays and ca. 47% for x-rays plus caffeine). This indicates that either the repair has not been completed or the apoptotic DNA fragmentation has been initiated (or both). The same treatment applied to x-irradiated (5 Gy) LY-R cells (G2 arrest, 4 h/Gy) has no radiosensitising effect, induces no apoptosis and does not alter the amount of DNA damage left unrepaired (ca. 28%). The results are compatible with the assumption that inhibition of the Atm-dependent homologous recombination repair by caffeine, brings differential effects in LY sublines because of the defect of the alternative DNA repair system (NHEJ) in LY-S cells. Received: 23 June 2000 / Accepted: 5 January 2001     

Ganglioside binding proteins of calf brain with ubiquitin-like N-terminals.

Two ganglioside-associated protein components I and II have been isolated from crude ganglioside preparations of calf brain by DEAE-Sephadex ion-exchange chromatography. Both components exhibited binding capacity in aqueous media for gangliosides of the 'ganglio' series but not for neutral glycosphingolipids (polyglycosylceramides) and only a low capacity for sialosylparagloboside. Each protein bound individual gangliosides with different efficiency. Upon prolonged incubation of component I with gangliosides, complexes with high (30:1) and low (6:1) glycolipid/protein molar ratios were formed. The latter but not the former complex was able to penetrate Sephadex G-200 beads. Both components inhibited plating efficiency of cultured mouse N2a neuroblastoma cells. The molecular masses of components I and II were determined by SDS/PAGE to be 11-12 kDa and 28 kDa, respectively. Carbohydrates (fucose, mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and some sialic acid) were found only in component II. When examined by reverse-phase HPLC each component separated into two major closely migrating peaks which were subsequently examined by Edman degradation. Amino acid sequences of the N-terminal portions of three of these peaks (one peak from component I and both peaks from component II) showed, as far as the sequences were established, identity with the sequence of ubiquitin. It is hypothesized that the proteins may be instrumental in intracellular trafficking of gangliosides.