Biodistribution of radioiodinated adenovirus fiber protein knob domain after intravenous injection in mice

The knob domains from the fiber proteins of adenovirus serotypes 2 and 12 were labeled with radioiodine and then injected into the bloodstreams of mice. Knob proteins with functional binding sites for the coxsackie and adenovirus receptor (CAR) were cleared rapidly from the circulation, with radioactivity appearing predominantly in the stomach, while knob mutants unable to bind to CAR remained in the blood circulation for a prolonged period. The clearance of radiolabeled wild-type knob from the blood was slowed by coinjecting an excess of unlabeled wild-type knob protein. An earlier study showed that (99m)Tc-labeled knob protein with intact CAR-binding activity also cleared rapidly from the blood circulation of mice, with radioactivity accumulating predominantly in the liver (K. R. Zinn et al., Gene Ther. 5:798-808, 1998). Together these results suggest that rapid clearance of knob protein from the blood results from specific binding to CAR in the liver and that the bound knob then enters a degradative pathway. The elevated levels of radioiodine in the stomach observed in our experiments are consistent with deiodination of labeled knob by dehalogenases in hepatocyte microsomes and uptake of the resultant free radioiodine by Na/I symporters in the gastric mucosa. Although CAR has been shown to localize in tight junctions of polarized epithelial cells, where it functions in intercellular adhesion, the results of our study suggest that a subset of CAR molecules in the liver is highly accessible to ligands in the blood and able to rapidly deliver bound ligand to an intracellular degradative compartment.     

Chemo- and stereoselective synthesis of benzocycloheptene and 1-benzoxepin derivatives as alpha-sympathomimetic and anorexigenic agents

The synthesis and pharmacological evaluation of cis- and trans-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ols 4a-c and 5a-c and cis- and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ols 4d-f and 5d-f were carried out. Chemo- and stereoselective synthesis of 5a-f was achieved by reduction of corresponding alpha-amino ketones 3a-f with LiAl(t-BuO)3H. cis-4-Amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 4d and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 5d exhibited marked anorexigenic activity in mice at a dose of LD50 800 and 500 mg/kg and ED50 75 and 55 mg/kg, respectively, while the analog cis-2,3-dihydroxy-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol 8 showed typical alpha-sympathomimetic activity.     

Structure of DNA sequence d-TGATCA by two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics

The 5' d-TpG 3' element is a part of DNA sequences involved in regulation of gene expression and is also a site for intercalation of several anticancer drugs. Solution conformation of DNA duplex d-TGATCA containing this element has been investigated by two-dimensional NMR spectroscopy. Using a total of 12 torsional angles and 121 distance constraints, structural refinement has been carried out by restrained molecular dynamics (rMDs) in vacuum up to 100 ps. The structure is characterized by a large positive roll at TpG/CpA base pair step and large negative propeller twist for AT and TA base pairs. The backbone torsional angle, gamma(O5'-C5'-C4'-C3'), of T1 residue adopts a trans-conformation which is corroborated by short intra nucleotide T1H6-T1H5' (3.7A) distance in nuclear overhauser effect spectroscopy (NOESY) spectra while the backbone torsional angle, beta(P-O5'-C5'-C4'), exists in trans as well as gauche state for T1 and C5 residues. There is evidence of significant flexibility of the sugar-phosphate backbone with rapid inter-conversion between two different conformers at TpG/CpA base pair step. The base sequence dependent variations and local structural heterogeneity have important implications in specific recognition of DNA by ligands.     

CTP:phosphocholine cytidylyltransferase inhibition by ceramide via PKC-alpha, p38 MAPK, cPLA2, and 5-lipoxygenase

In a companion paper (Vivekananda J, Smith D, and King RJ. Am J Physiol Lung Cell Mol Physiol 281: L98-L107, 2001), we demonstrated that tumor necrosis factor (TNF)-alpha inhibited the activity of CTP:phosphocholine cytidylyltransferase (CT), the rate-limiting enzyme in the de novo synthesis of phosphatidylcholine (PC), and that its actions were likely exerted through a metabolite of sphingomyelin. In this paper, we explore the signaling pathway employed by TNF-alpha using C2 ceramide as a cell-penetrating sphingolipid representative of the metabolites induced by TNF-alpha. We found that in H441 cells, as reported in other cell types, cytosolic phospholipase A2 (cPLA2) is activated by TNF-alpha. We also observed that the inhibiting action of C2 ceramide on CT requires protein kinase C-alpha, p38 mitogen-activated protein kinase, and cPLA2. The actions of C2 ceramide on CT activity can be duplicated by adding 2 microM lysoPC to these cells. Furthermore, we found that the effects of C2 ceramide are dependent on 5-lipoxygenase but that cyclooxygenase II is unimportant. We hypothesize that CT activity is inhibited by the lysoPC generated as a consequence of the activation of cPLA2 by protein kinase C-alpha and p38 mitogen-activated protein kinase. The other product of the activation of cPLA2, arachidonic acid, is a substrate for the synthesis of leukotrienes, which raise intracellular Ca2+ levels and complete the activation of cPLA2.     

Activity of allelic variants of Pi class human glutathione S-transferase toward chlorambucil

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil, is often limited by the emergence of drug resistant tumor cells. Increased glutathione (GSH) conjugation (inactivation) of alkylating anticancer drugs or their activated metabolites due to overexpression of the Pi class GSH S-transferase (hGSTP1-1) is believed to be an important mechanism in tumor cell resistance to alkylating agents. Interestingly, the hGSTP1 locus is polymorphic in human populations and involves amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine). Here, we report that the allelic variants of hGSTP1-1 significantly differ in their efficiency in catalyzing the GSH conjugation of chlorambucil. Catalytic efficiency of the hGSTP1-1(I104,A113) isoform toward chlorambucil was approximately 2.5-, 7.5- and 15-fold higher compared with I104,V113, V104,A113 and V104,V113 variants of hGSTP1-1, respectively. The results of the present study suggest that hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to some alkylating agents.     

PKC, p42/p44 MAPK, and p38 MAPK are required for HGF-induced proliferation of H441 cells

In this paper, we studied the signaling pathway used by hepatocyte growth factor/scatter factor (HGF) to stimulate mitosis. We show, using H441 cells, that 1) HGF activates membrane-associated protein kinase C (PKC); the activity is transient and peaks within 30 min; 2) HGF activates p42/p44 and p38 mitogen-activated protein kinases (MAPKs); maximum activity in both is within 10 min; and 3) the activation of neither p38 nor p42/p44 MAPK is dependent on PKC, indicating that HGF uses separate and nonintersecting pathways to activate these two classes of kinase. However, phorbol 12-myristate 13-acetate also activates both MAPKs as well as PKC, but this activation is abolished in cells pretreated with the PKC inhibitor GF-109203X. HGF was found to significantly increase [(3)H]thymidine incorporation within 5 h; peak thymidine incorporation was observed at 16 h. However, when cells were pretreated with inhibitors of p42/p44 (PD-98059), p38 (SB-203580), or PKC (GF-109203X, G?-6983, or myristoylated inhibitor peptide(19-27)), HGF-induced thymidine uptake was diminished in a dose-dependent manner. Taken together, these results demonstrate that HGF activates PKC and both MAPKs simultaneously through parallel pathways and that the activation of the MAPKs does not depend on PKC. However, p38 and p42/p44 MAPKs and PKC may all be essential for HGF-induced proliferation of H441 cells.