Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine

The activity of thymidylate synthase (TS) purified in our laboratory from Lactobacillus leichmannii was inhibited by pergularinine (PGL) and tylophorinidine (TPD) and deoxytubulosine (DTB) isolated from the Indian medicinal plants Pergularia pallida and Alangium lamarckii respectively. Cytotoxicity studies showed that cell growth of L. leichmannii was inhibited (IC50 = 40-45 microM) by all the three alkaloids, the concentrations > 80-90 microM resulting in complete loss of the enzyme activity. Ki values of the enzyme calculated from Lineweaver-Burk and Dixon plots for PGL, TPD and DTB were 10 x 10(-6) M, 9 x 10(-6) M and 7 x 10(-6) M respectively. These are typed as 'non-competitive' inhibitors of TS. All the three alkaloids inhibited (IC50 = 50 microM) the elevated TS activity of leukocytes in cancer patients with clinically diagnosed chronic myelocytic leukemia (n = 10), acute lymphocytic leukemia (n = 8) and metastatic solid tumours (n = 3).     

Free radical scavenging ability and antioxidant efficiency of curcumin and its substituted analogue

Free radical reactions of curcumin and its ethoxy substituted derivative (C1) 1,7-bis-(4-hydroxy-3-ethoxy phenyl)-1,6-heptadiene-3,5-dione have been studied using a pulse radiolysis technique in homogeneous aqueous-organic solutions like acetonitrile-water and isopropanol-water mixtures, as well as in neutral TX-100 and cationic CTAB micellar solutions. The phenoxyl radicals of curcumin or C1 were generated by one-electron transfer to several oxidants like N(3)(.), Br(2)(-.), CCl(3)O(2)(.), glutathione radicals which exhibit absorption from a 300-600-nm wavelength region with the maximum at 490-500 nm. Other important properties of the phenoxyl radicals such as extinction coefficient, radical lifetime and their formation and decay rate constants were also determined in these systems. The antioxidant property of curcumin and C1 were estimated in terms of their ability to inhibit the lipid peroxidation in liposomes and also in terms of trolox equivalent antioxidant capacity (TEAC). The results were compared with alpha-tocopherol.     

Cyclin D1 genetic heterozygosity regulates colonic epithelial cell differentiation and tumor number in ApcMin mice

Constitutive β-catenin/Tcf activity, the primary transforming events in colorectal carcinoma, occurs through induction of the Wnt pathway or APC gene mutations that cause familial adenomatous polyposis. Mice carrying Apc mutations in their germ line (Apc^Min) develop intestinal adenomas. Here, the crossing of Apc^Min with cyclin D1^−/− mice reduced the intestinal tumor number in animals genetically heterozygous or nullizygous for cyclin D1. Decreased tumor number in the duodenum, intestines, and colons of Apc^Min/cyclin D1^+/− mice correlated with reduced cellular proliferation and increased differentiation. Cyclin D1 deficiency reduced DNA synthesis and induced differentiation of colonic epithelial cells harboring mutant APC but not wild-type APC cells in vivo. In previous studies, the complete loss of cyclin D1 through homozygous genetic deletion conveyed breast tumor resistance. The protection of mice, genetically predisposed to intestinal tumorigenesis, through cyclin D1 heterozygosity suggests that modalities that reduce cyclin D1 abundance could provide chemoprotection.     

PAM14, a novel MRG- and Rb-associated protein, is not required for development and T-cell function in mice

PAM14 has been found to associate in complexes with the MORF4/MRG family of proteins as well as Rb, the tumor suppressor protein. This suggested that it might be involved in cell growth, immortalization, and/or senescence. To elucidate the in vivo function of PAM14, we characterized the expression pattern of mouse Pam14 and generated PAM14-deficient (Pam14(-/-)) mice. Pam14 was widely expressed in all mouse tissues and as early as 7 days during embryonic development. Despite this ubiquitous expression in wild-type mice, Pam14(-/-) mice were healthy and fertile. Response to mitogenic stimulation and production of interleukin-2 were the same in stimulated splenic T cells from Pam14(-/-) mice as in control littermates. Cell growth rates of mouse embryonic fibroblasts (MEFs) from all three genotypes were the same, and immortalized cells were obtained from all cell cultures during continuous culture. There was also no difference in expression of growth-related genes in response to serum stimulation in the null versus control MEFs. These data demonstrate that PAM14 is not essential for normal mouse development and cell cycle control. PAM14 likely acts as an adaptor protein in nucleoprotein complexes and is probably compensated for by another functionally redundant protein(s).     

Unidirectional translocation from recognition site and a necessary interaction with DNA end for cleavage by Type III restriction enzyme

Type III restriction enzymes have been demonstrated to require two unmethylated asymmetric recognition sites oriented head-to-head to elicit double-strand break 25–27 bp downstream of one of the two sites. The proposed DNA cleavage mechanism involves ATP-dependent DNA translocation. The sequence context of the recognition site was suggested to influence the site of DNA cleavage by the enzyme. In this investigation, we demonstrate that the cleavage site of the R.EcoP15I restriction enzyme does not depend on the sequence context of the recognition site. Strikingly, this study demonstrates that the enzyme can cleave linear DNA having either recognition sites in the same orientation or a single recognition site. Cleavage occurs predominantly at a site proximal to the DNA end in the case of multiple site substrates. Such cleavage can be abolished by the binding of Lac repressor downstream (3′ side) but not upstream (5′ side) of the recognition site. Binding of HU protein has also been observed to interfere with R.EcoP15I cleavage activity. In accordance with a mechanism requiring two enzyme molecules cooperating to elicit double-strand break on DNA, our results convincingly demonstrate that the enzyme translocates on DNA in a 5′ to 3′ direction from its recognition site and indicate a switch in the direction of enzyme motion at the DNA ends. This study demonstrates a new facet in the mode of action of these restriction enzymes.     

Liposome-stabilized oil-in-water emulsions as adjuvants: increased emulsion stability promotes induction of cytotoxic T lymphocytes against an HIV envelope antigen

Protective or therapeutic immunity against HIV infection is currently believed to require both antibody and CTL responses against the envelope protein. In the present study, the adjuvant activity of a unique oil-in-water emulsion, in which liposomes containing lipid A (LA) and encapsulated antigen served as the emulsifying agent, was examined in mice using oligomeric gp140 (ogp140) derived from the HIV-1 envelope as the antigen. Emulsions rendered either highly stable or unstable by altering the ratio of liposomes to oil were used to examine the effect of stability of the emulsion on adjuvant activity. Stable and unstable emulsions had similar potencies for inducing both IgG antibodies to ogp140 and antigen-specific T-lymphocyte proliferation. Stable emulsions, but not unstable emulsions, induced antigen-specific CTL responses, possibly because of the depot effect of the stable emulsions. Furthermore, stable emulsions induced lower IgG2a/IgG1 ratios than the unstable emulsions. We conclude that stable liposomal oil-in-water emulsions provide an effective means of obtaining both antibody and CTL responses against an HIV envelope antigen.     

Cloning and characterization of Kin5, a novel Tetrahymena ciliary kinesin II

Two Tetrahymena kinesin-like proteins (klps) of the kinesin II subfamily, Kin1 and Kin2, were first identified by Brown et al. [1999: Mol Biol Cell 10: 3081-3096] and shown to be involved in ciliary morphogenesis probably as molecular motors in intraciliary transport (ICT). Using Tetrahymena genomic DNA as a template, we cloned Kin5, another kinesin II subfamily member. Kin5 is upregulated upon deciliation, suggesting that Kin5 is a ciliary protein. Kin5 is most closely related to Osm3, a Caenorhabditis elegans kinesin II; Osm3 and Kin5 have a 56% identity, which rises to 60.4% in the motor domain and a 45% identity in a 60 amino acid region of the C-terminal FERM (4.1, Ezrin, Radixin, Moesin) domain, not present in Kin1 or Kin2, which we hypothesize to be a critical domain either for dimerization or for cargo recognition in ICT. An antibody to a peptide sequence from the tail region of Kin5 localizes in a punctate pattern along the ciliary axoneme, colocalizing with an antibody to the raft protein IFT139. These findings suggest that Kin5 is an ICT motor like Osm3. Osm3 orthologs apparently transport membrane proteins and Kin5 may be the homodimeric kinesin II that performs this function in Tetrahymena cilia.