Association of functionally important polymorphisms in cytochrome P4501B1 with lung cancer

In the present study, genotype and haplotype frequencies of four polymorphisms of cytochrome P450 1B1 (CYP1B1) that cause amino acid changes (Arg-Gly at codon 48, Ala-Ser at codon 119, Leu-Val at 432 and Asn-Ser at codon 453) were studied in 200 patients suffering from lung cancer and equal number of controls. A significant difference was observed for the distribution of variant genotypes of CYP1B1Arg48Gly and Ala119Ser polymorphisms (CYP1B1*2) in cases when compared to the controls. No significant difference was observed for the distribution of variant genotypes of CYP1B1Leu432Val (CYP1B1*3) and CYP1B1Asn453Ser (CYP1B1*4) polymorphism. When the four SNPs were analyzed using a haplotype approach, SNPs at codon 48 (Arg48Gly) and codon 119 (Ala119Ser) exhibited complete linkage disequilibrium (LD) in all the cases and controls. Significant differences in the distribution of the three haplotypes (G-T-C-A, G-T-G-A and G-T-C-G) were observed in the cases when compared to controls. Tobacco use in the form of smoking as well as chewing was found to significantly increase the risk of lung cancer in patients by interacting with CYP1B1Ala119Ser genotypes demonstrating the role of gene-environment interaction in lung cancer. Further, the risk of lung cancer increased several fold in the patients carrying the genotype combinations of CYP1B1Ala119Ser and CYP1B1Leu432Val with GSTM1, a phase II enzyme suggesting the importance of gene-gene interactions in enhancing the susceptibility to lung cancer.     

Roles of cell and microvillus deformation and receptor-ligand binding kinetics in cell rolling

Polymorphonuclear leukocyte (PMN) recruitment to sites of inflammation is initiated by selectin-mediated PMN tethering and rolling on activated endothelium under flow. Cell rolling is modulated by bulk cell deformation (mesoscale), microvillus deformability (microscale), and receptor-ligand binding kinetics (nanoscale). Selectin-ligand bonds exhibit a catch-slip bond behavior, and their dissociation is governed not only by the force but also by the force history. Whereas previous theoretical models have studied the significance of these three "length scales" in isolation, how their interplay affects cell rolling has yet to be resolved. We therefore developed a three-dimensional computational model that integrates the aforementioned length scales to delineate their relative contributions to PMN rolling. Our simulations predict that the catch-slip bond behavior and to a lesser extent bulk cell deformation are responsible for the shear threshold phenomenon. Cells bearing deformable rather than rigid microvilli roll slower only at high P-selectin site densities and elevated levels of shear (>or=400 s(-1)). The more compliant cells (membrane stiffness=1.2 dyn/cm) rolled slower than cells with a membrane stiffness of 3.0 dyn/cm at shear rates >50 s(-1). In summary, our model demonstrates that cell rolling over a ligand-coated surface is a highly coordinated process characterized by a complex interplay between forces acting on three distinct length scales.     

Myristoylation and Membrane Binding Regulate c-Src Stability and Kinase Activity

Myristoylation is critical for membrane association of Src kinases, but a role for myristate in regulating other aspects of Src biology has not been explored. In the c-Abl tyrosine kinase, myristate binds within a hydrophobic pocket at the base of the kinase domain and latches the protein into an autoinhibitory conformation. A similar pocket has been predicted to exist in c-Src, raising the possibility that Src might also be regulated by myristoylation. Here we show that in contrast to the case for c-Abl, myristoylation exerts a positive effect on c-Src kinase activity. We also demonstrate that myristoylation and membrane binding regulate c-Src ubiquitination and degradation. Nonmyristoylated c-Src exhibited reduced kinase activity but had enhanced stability compared to myristoylated c-Src. We then mutated critical residues in the predicted myristate binding pocket of c-Src. Mutation of L360 and/or E486 had no effect on c-Src membrane binding or localization. However, constructs containing a T456A mutation were partially released from the membrane, suggesting that mutagenesis could induce c-Src to undergo an artificial myristoyl switch. All of the pocket mutants exhibited decreased kinase activity. We concluded that myristoylation and the pocket residues regulate c-Src, but in a manner very different from that for c-Abl.Src family kinases (SFKs) are nonreceptor tyrosine kinases that act as key mediators of cellular signal transduction (12). The nine SFK members, Src, Yes, Fyn, Hck, Lck, Lyn, Blk, Fgr, and Yrk, play crucial roles in cellular proliferation, survival, migration, and growth factor and cytokine stimulation pathways (26, 39, 56). All SFKs share a similar domain arrangement, consisting of SH3, SH2, and kinase (SH1) domains as well as a unique domain and a membrane-targeting SH4 region at the N terminus (11). Crystal structures have shown that the catalytic activity of SFKs is tightly regulated by autoinhibition. The SH3 domain binds to a polyproline region in the linker between the SH2 and kinase domains, and the SH2 domain binds to a phosphotyrosine residue (Tyr527 in avian c-Src) near the C terminus. Kinase activation can be achieved by displacing one or all of the autoinhibitory interactions (52, 59, 60).All SFKs are myristoylated at the N terminus (47). Myristoylation occurs cotranslationally and is catalyzed by the enzyme N-myristoyl transferase (NMT) (19). The 14-carbon saturated fatty acid myristate is covalently attached to the N-terminal glycine residue via an amide bond, making myristoylation an essentially irreversible modification (44, 48, 49). Myristoylation is necessary but not sufficient to anchor a protein to the membrane, and membrane binding of myristoylated proteins requires a second signal. For Src, the second signal is a polybasic cluster of amino acids that interacts with acidic phospholipids on the inner leaflet of the membrane bilayer (34, 35, 44, 46, 53). Nearly all other SFKs are instead modified by attachment of the 16-carbon saturated fatty acid palmitate to cysteine residues 3 and 5 or 6 at the N terminus (48). Myristoylation and palmitoylation together form a “dual signal” motif that targets SFKs to membranes.Membrane binding is crucial for cellular functions mediated by Src and other SFKs. Nonmyristoylated forms of Src are cytoplasmic and cannot induce cellular transformation (14, 28, 29, 33). Membrane localization of c-Src has been shown to be important for dephosphorylation of Tyr527 and for mitotic activation of c-Src kinase activity (8), presumably because the phosphatase that acts on Tyr527 is membrane bound. Myristoylation has also been proposed to play a role in regulating nuclear transport of c-Src (17).For some myristoylated proteins, the myristate moiety can exist in two different conformational states, either sequestered inside a hydrophobic pocket within the protein or exposed and available for membrane binding (44, 48). Binding to a ligand or another protein can cause a switch from one state to another, resulting in membrane association or dissociation. “Myristoyl switch” mechanisms have been identified in a variety of myristoylated proteins, including recoverin and HIV-1 Gag (4-6, 43, 45, 55). In the c-Abl tyrosine kinase, a “myristoyl phosphotyrosine” switch is operative. Myristate binds within a hydrophobic pocket at the base of the c-Abl kinase domain, docking the SH2 domain against the kinase domain in such a way that it prevents activation of the kinase by phosphotyrosine ligands (22, 36). A similar pocket is predicted to exist at the base of the c-Src kinase domain, raising the possibility that c-Src in the autoinhibited form might be capable of binding its own N-terminal myristate group in a manner similar to that of c-Abl (16). To date, only nonmyristoylated, N-terminally truncated forms of c-Src have been crystallized, and the position of the myristate within the full-length c-Src protein is not known. A recent study provided support for the existence of a potential myristate binding pocket within c-Src (16). Exogenous addition of myristate to the Tyr527-phosphorylated form of nonmyristoylated c-Src induced chemical shift changes in the nuclear magnetic resonance (NMR) spectra for both the protein and the fatty acid. However, the site of myristate binding was not determined. Thus, it is still not known how or if myristate regulates c-Src kinase activity and whether the predicted myristate binding pocket functions within c-Src in a manner similar to that of c-Abl.In this study, we directly analyzed the role of myristoylation in regulating c-Src kinase activity and tested whether residues in the predicted myristate binding pocket contribute to myristate binding and/or c-Src activity. Here we show that myristoylation plays a positive role in regulating c-Src kinase activity. In contrast to c-Abl, nonmyristoylated c-Src exhibits reduced kinase activity both in vitro and in cells. We also made the surprising finding that the myristoylation status of c-Src determines its intracellular stability by regulating c-Src ubiquitination and degradation of the E3 ligase Cbl. Lastly, we analyzed the role of the predicted myristate binding pocket at the base of the c-Src kinase domain. Mutations in the pocket region resulted in decreased kinase activity and, with the exception of one mutation (Thr456Ala), had no effect on membrane binding of c-Src. We concluded that c-Src kinase activity is regulated by myristoylation, but in a different manner from that of c-Abl, and that a “myristoyl switch” is unlikely to be operative within c-Src.     

Identification and synthesis of a unique thiocarbazate cathepsin L inhibitor

Library samples containing 2,5-disubstituted oxadiazoles were identified as potent hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) directed at discovering inhibitors of cathepsin L. However, when synthesized in pure form, the putative actives were found to be devoid of biological activity. Analyses by LC-MS of original library samples indicated the presence of a number of impurities, in addition to the oxadiazoles. Synthesis and bioassay of the probable impurities led to the identification of a thiocarbazate that likely originated via ring opening of the oxadiazole. Previously unknown, thiocarbazates (-)-11 and (-)-12 were independently synthesized as single enantiomers and found to inhibit cathepsin L in the low nanomolar range.     

Synthesis, cellular transport, and activity of polyamidoamine dendrimer-methylprednisolone conjugates

Dendrimers have emerged as promising multifunctional nanomaterials for drug delivery due to their well-defined size and tailorability. We compare two schemes to obtain methylprednisolone (MP)-polyamidoamine dendrimer (PAMAM-G4-OH) conjugate. Glutaric acid (GA) was used as a spacer to facilitate the conjugation. In scheme A, PAMAM-G4-OH was first coupled to GA and then further conjugated with MP to obtain PAMAM-G4-GA-MP conjugates. This scheme yields a lower conjugation ratio of MP, presumably because of lower reactivity and steric hindrance for the steroid at the crowded dendrimer periphery. In scheme B, this steric hindrance was overcome by first preparing the MP-GA conjugate, which was then coupled to the PAMAM-G4-OH dendrimer. The (1)H NMR spectrum of the conjugate from scheme B indicates a conjugation of 12 molecules of MP with the dendrimer, corresponding to a payload of 32 wt %. In addition, conjugates were further fluorescent-labeled with fluoroisothiocynate (FITC) to evaluate the dynamics of cellular entry. Flow cytometry and UV/visible spectroscopic analysis showed that the conjugate is rapidly taken up inside the cell. Fluorescence and confocal microscopy images on A549 human lung epithelial carcinoma cells treated with conjugates show that the conjugate is mostly localized in cytosol. MP-GA-dendrimer conjugate showed comparable pharmacological activity to free MP, as measured by inhibition of prostaglandin secretion. These conjugates can potentially be further conjugated with a targeting moiety to deliver the drugs to specific cells in vivo.     

Regulation of endogenous glucose production after a mixed meal in type 2 diabetes

The extent and time course of suppression of endogenous glucose production (EGP) in type 2 diabetes after a mixed meal have been determined using a new tracer methodology. Groups of age-, sex-, and weight-matched normal controls (n = 8) and diet-controlled type 2 diabetic subjects (n = 8) were studied after ingesting a standard mixed meal (550 kcal; 67% carbohydrate, 19% fat, 14% protein). There was an early insulin increment in both groups such that, by 20 min, plasma insulin levels were 266 +/- 54 and 190 +/- 53 pmol/l, respectively. EGP was similar basally [2.55 +/- 0.12 mg x kg(-1) x min(-1) in control subjects vs. 2.92 +/- 0.16 mg x kg(-1) x min(-1) in the patients (P = 0.09)]. After glucose ingestion, EGP declined rapidly in both groups to approximately 50% of basal within 30 min of the meal. Despite the initial rapid decrease, the EGP was significantly greater in the diabetic group at 60 min (1.75 +/- 0.12 vs. 1.05 +/- 0.14 mg x kg(-1) x min(-1); P < 0.01) and did not reach nadir until 210 min (0.96 +/- 0.17 mg x kg(-1) x min(-1)). Between 60 and 240 min, EGP was 47% higher in the diabetic group (0.89 +/- 0.09 vs. 1.31 +/- 0.13 mg x kg(-1) x min(-1), P < 0.02). These data quantitate the initial rapid suppression of EGP after a mixed meal in type 2 diabetes and the contribution of continuing excess glucose production to subsequent hyperglycemia.     

Improvement in ductility of chitosan through blending and copolymerization with PEG: FTIR investigation of molecular interactions

Chitosan is an important biomaterial used widely in medical applications. One of the key concerns about its use is the fragile nature of chitosan films. By comparing the component molecular interactions using FTIR, this study attempts to understand how the ductility of chitosan can be improved by blending and copolymerizing with poly(ethylene glycol) (PEG). An improvement in ductility was obtained for all compositions of blend as manifested by a decrease in modulus and an increase in strain at break. For comparable PEG composition (approximately 30%), the properties of the solution-cast blend were better than those of the grafted copolymer. Therefore, blending may be a more efficient way to improve ductility of chitosan. FTIR characterization of the materials revealed subtle decreases in molecular interactions upon annealing the partially miscible blend. These may not be apparent in DSC or X-ray diffraction, yet they play a key role in the mechanical behavior. It appears that in the case of the graft copolymer the improvement in the properties comes from suppression of the crystallinity of each component and not from component interactions. On the other hand, in the blend, the improvement appears to come predominantly from the "well-dispersed", "kinetically trapped" phase morphology and from the intermolecular interactions. Therefore, annealing the blend leads to decreased intermolecular interactions, phase coarsening, and deterioration in properties.