The presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation.

8-Oxoguanine (8-oxoG), induced by reactive oxygen species (ROS) and ionizing radiation, is arguably the most important mutagenic lesion in DNA. This oxidized base, because of its mispairing with A, induces GC-->TA transversion mutations often observed spontaneously in tumor cells. The human cDNA encoding the repair enzyme 8-oxoG-DNA glycosylase (OGG-1) has recently been cloned, however, its activity was never detected in cells. Here we show that the apparent lack of this activity could be due to the presence of an 8-oxoG-specific DNA binding protein. Moreover, we demonstrate the presence of two antigenically distinct OGG activities with an identical reaction mechanism in human cell (HeLa) extracts. The 38 kDa OGG-1, identical to the cloned enzyme, cleaves 8-oxoG when paired with cytosine, thymine and guanine but not adenine in DNA. In contrast, the newly discovered 36 kDa OGG-2 prefers 8-oxoG paired with G and A. We propose that OGG-1 and OGG-2 have distinct antimutagenic functions in vivo . OGG-1 prevents mutation by removing 8-oxoG formed in DNA in situ and paired with C, while OGG-2 removes 8-oxoG that is incorporated opposite A in DNA from ROS-induced 8-oxodGTP. We predict that OGG-2 specifically removes such 8-oxoG residues only from the nascent strand, possibly by utilizing the same mechanism as the DNA mismatch repair pathway.     

A Zn(II)-translocating P-type ATPase from Proteus mirabilis.

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis gene was expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.     

Conformational flexibilities in malformin A

The linked-atom-least-squares (LALS) technique has been applied to generate exactly cyclized and stereochemically satisfactory conformations of the cyclic pentapeptide, malformin A, which contains an intramolecular disulfide bridge across a D-Cys—D-Cys linkage. Consistent with theoretical, ir, and x-ray evidence from studies on analogs of the LKiysteinyl—L-cysteine disulfide, it is shown that the peptide bond across the S? S bridge in malformin A can retain a cis configuration. The two all-trans structures proposed earlier by A.E. Tonelli [(1978) Biopolymers 17, 1175–1179] from solution nmr data have also been analyzed. Both P-(χ^s ～ 90°) and M-(χ^s ～ ?90°)-type helicity of the S? S bridge are found to be accessible in both the trans and the cis models in which the respective conformations of the homodetic rings are essentially preserved. The details of six different conformational states and their relative energies have been evaluated and compared. Our findings, which suggest that a variety of conformational states are accessible to malformin A, are compatible with other published results from solution studies. On the basis of hydrogen-bonding interactions, a model is proposed to explain how malformin A might be inactivated specifically by L -Hyp, and not by L -Pro or other amino acids, as has been observed from in vivo investigations [Buckhout, T.J. & Curtis, R.W. (1976) Nature 260 , 435–436]     

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.     

An isoflavone glycoside from the heartwood of Pterocarpus marsupium

A new isoflavone, 5,4′-dimethoxy-8-methylisoflavone, has been identified from the heartwood of Pterocarpus marsupium.     

Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway

Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.     

Taxol allosterically alters the dynamics of the tubulin dimer and increases the flexibility of microtubules

Taxol is a commonly used antitumor agent that hyperstabilizes microtubules and prevents cell division. The interaction of Taxol with tubulin and the microtubule has been studied through a wide array of experimental techniques; however, the exact molecular mechanism by which Taxol stabilizes microtubules has remained elusive. In this study, through the use of large-scale molecular simulations, we show that Taxol affects the interactions between the M and H1-S2 loops of adjacent tubulin dimers leading to more stable interprotofilament interactions. More importantly, we demonstrate that Taxol binding leads to a significant increase in the dynamics and flexibility of the portion of β-tubulin that surrounds the bound nucleotide and makes contact with the α-monomer of the next dimer in the protofilament. We conclude that this increase in flexibility allows the microtubule to counteract the conformational changes induced by nucleotide hydrolysis and keeps the protofilaments in a straight conformation, resulting in a stable microtubule.     

Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation

Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.