Activation of human MutS homologs by 8-oxo-guanine DNA damage.

The DNA lesion 8-oxo-guanine (8-oxo-G) is a highly mutagenic product of the interaction between reactive oxygen species and DNA. To maintain genomic integrity, cells have evolved mechanisms capable of removing this frequently arising oxidative lesion. Mismatch repair (MMR) appears to be one pathway associated with the repair of 8-oxo-G lesions (DeWeese, T. L., Shipman, J. M., Larrier, N. A., Buckley, N. M., Kidd, L. R., Groopman, J. D., Cutler, R. G., te Riele, H., and Nelson, W. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11915-11920; Ni, T. T., Marsischky, G. T., and Kolodner, R. D. (1999) Mol. Cell 4, 439-444). Here we report the effect of double-stranded DNA oligonucleotides containing a single 8-oxo-G on the DNA binding affinity, ATPase, and ADP right arrow ATP exchange activities of hMSH2-hMSH6 and hMSH2-hMSH3. We found that hMSH2-hMSH6 binds the oligonucleotide DNA substrates with the following affinities: 8-oxo-G/T > 8-oxo-G/G > 8-oxo-G/A > 8-oxo-G/C approximately G/C. A similar trend was observed for DNA-stimulated ATPase and ADP --> ATP exchange activities of hMSH2-hMSH6. In contrast, hMSH2-hMSH3 did not appear to bind any of the 8-oxo-G containing DNA substrates nor was there enhanced ATPase or ADP --> ATP exchange activities. These results suggest that only hMSH2-hMSH6 is activated by recognition of 8-oxo-G lesions. Our data are consistent with the notion that post-replication MMR only participates in the repair of mismatched 8-oxo-G lesions.     

The Role of Phosphometabolites in Cell Proliferation, Energy Metabolism, and Tumor Therapy

A common characteristic of tumor cells is the constant overexpression of glycolytic and glutaminolytic enzymes. In tumor cells the hyperactive hexokinase and the partly inactive pyruvate kinase lead to an expansion of all phosphometabolites from glucose 6-phosphate to phosphoenolpyruvate. In addition to the glycolytic phosphometabolites, synthesis of their metabolic derivatives such as P-ribose-PP, NADH, NADPH, UTP, CTP, and UDP-N-acetyl glucosamine is also enhanced during cell proliferation. Another phosphometabolite derived from P-ribose-PP, AMP, inhibits cell proliferation. The accumulation of AMP inhibits both P-ribose-PP-synthetase and the increase in concentration of phosphometabolites derived from P-ribose-PP. In cells with low glycerol 3-phosphate and malate-aspartate shuttle capacities the inhibition of the lactate dehydrogenase by low NADH levels leads to an inhibition of glycolytic ATP production. Several tumor-therapeutic drugs reduce NAD and NADH levels, thereby inhibiting glycolytic energy production. The role of AMP, NADH, and NADPH levels in the success of chemotherapeutic treatment is discussed.     

Synthesis and hydrolysis of oligodeoxyribonucleotides containing 2-aminopurine.

A new method is reported for the synthesis of oligodeoxyribonucleotides containing 2-aminopurine residues at selected sites. This method involves protection of the 2-aminopurine ribonucleoside, reduction to the deoxyribonucleoside and standard preparation of the 5'-0- (4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyl)-N,N- diisopropylphosphoramidite. The 2-aminopurine phosphoramidite prepared by this method couples with high efficiency and is stable under standard automated synthesis conditions. The presence and location of the 2-aminopurine residue is easily verified by treatment of the oligodeoxyribonucleotide with hot piperidine. The mechanism for selective hydrolysis of the 2-aminopurine residue in alkaline solution is predominantly direct cleave of the glycosidic bond.     

The development ofChlorella vulgaris cells exposed to cadmium at successive stages of their life cycle

The effect of CdCl₂ in a concentration range 0.01-10.0 g m^-3 on the growth ofChlorella vulgaris under synchronous cultivation conditions was determined. The general biological activity, the growth multiplication factor, the cell size and shape and intracellular arrangement showed disturbances of synchronization that depended on Cd²⁺ concentration. The highest inhibition of all mentioned parameters was observed when Cd²⁺ was administered after the second hour of synchronous cultivation, whereas the administration after 6 or 8 h did not induce any significant effect.     

The binding of prototype lexitropsins to the minor groove of DNA: quantum chemical studies.

Ab initio calculations (Hartree-Fock) using the 6-31 G basis set have been performed on two prototype lexitropsins or information-reading molecules. The latter are DNA minor groove binding agents related to the A.T recognizing netropsin in which each of the two N-methylpyrrole moieties is replaced in turn by 1-methylimidazole and which thereby confers the property of recognizing G.C sites.Ab initio treatment was possible by examining composities of separate non-conjugated segments of the molecules. Geometry optimized conformations, energies and distribution of electrostatic charges within the molecules were derived. The ab initio derived parameters of the geometry optimized conformations of these lexitropsins were used to interpret their interaction with different sequences within the minor groove of B-DNA.     

Translocation of protein kinase C is not required to inhibit the antigen-induced increase of cytosolic calcium in a mast cell line

Cross-linking of receptor bound IgE antibodies by multivalent antigen (DNP8-BSA) on PB-3c cells leads to an increase of cytosolic calcium ((Ca2+)i). Active tumor promoting phorbol esters and teleocidin which specifically activate the phospholipid Ca2+-sensitive protein kinase (PKC), inhibited the antigen-mediated rise in (Ca2+)i and induced a time and dose-dependent translocation of cytosolic PKC to membranes of the PB-3c cells as determined by enzyme activity or immunoblotting using a polyclonal anti-PKC antibody. This TPA concentration did not affect the subcellular distribution of PKC, although 1 nM of 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited to 50% the antigen-mediated increase in (Ca2+)i. The concentration of TPA required to induce a half-maximal subcellular redistribution of immunodetectable PKC activity was an order of magnitude greater than the half-maximal dose required to inhibit the antigen-mediated increase in (Ca2+)i. These data demonstrate that the TPA-dependent activation of PKC is not directly coupled to its translocation to membranes.     

Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B(1)R) and bradykinin receptor 2 (B(2)R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B(2)R and B(1)R proteins are expressed in the epithelial cells of normal and IBD intestines. B(1)R protein is visualized in macrophages at the center of granulomas in CD. B(2)R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B(1)R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B(1)R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B(1)R mRNA is significantly higher than B(2)R mRNA. However, in inactive UC patients, the B(1)R and B(2)R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B(1)R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.     

Reduced concentrations of arginine vasopressin and MHPG in lumbar CSF of patients with Korsakoff's psychosis

The concentrations of arginine vasopressin (AVP), somatostatin (SS), and the primary brain metabolites of norepinephrine (MHPG), serotonin (5-HIAA), and dopamine (HVA) were measured in samples of lumbar CSF obtained from ten amnesics with Korsakoff's psychosis, four patients with a history of Korsakoff's psychosis who had recovered from the amnesic symptoms of this disease, and control subjects. Significant deficits were observed in the amnesic group for AVP and MHPG, but not for the other substances measured. Subjects who had recovered from the amnesic symptoms of Korsakoff's psychosis had increased concentrations of AVP and MHPG, but not of SS or the other monoamine metabolites.