Interaction of liposome-encapsulated cisplatin with biomolecules

We prepared liposomes by hydrating 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid with aqueous solutions of three "probe" molecules-cis-diamminedichloroplatinum(II) (cis-[Pt(II)(NH(3))(2)Cl(2)], cisplatin), guanosine 5'-monophosphate (5'-GMP), and 9-ethylguanine (9-EtG)-in phosphate-buffered saline as well as N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid buffer. The positively charged hydrolysis product of cisplatin, [Pt(II)(NH(3))(2)Cl(H(2)O)](+), is in the inner core of the liposomes and negatively charged 5'-GMP embeds in the lipid bilayer of liposomes. In the presence of cisplatin, the size of the liposomes remains unchanged, and for 5'-GMP-embedded liposomes the size increases significantly compared with that of empty or control liposomes. In contrast, the neutral biomolecule 9-EtG was found to be dispersed in the exterior bulk water and the size of the liposomes remained the same as that of empty or control liposomes. When cisplatin-containing liposomes mix with 5'-GMP-embedded liposomes or liposomes with 9-EtG, the N7 nitrogen atom of 5'-GMP or 9-EtG binds the cisplatin, thus replacing the "leaving groups" and forming a bisadduct. After 48?h of mixing, the size of the liposomes changes for the mixture of 5'-GMP-embedded liposomes and cisplatin-containing liposomes. We used (1)H and (31)P NMR spectroscopic techniques to monitor incorporation or association of cisplatin and biomolecules with liposomes and their subsequent reactions with each other. The dynamic light scattering technique provided the size distribution of the liposomes in the presence and absence of probe molecules.     

Evolution of CRISPs associated with toxicoferan-reptilian venom and mammalian reproduction

Cysteine-rich secretory proteins (CRISPs) are glycoproteins found exclusively in vertebrates and have broad diversified functions. They are hypothesized to play important roles in mammalian reproduction and in reptilian venom, where they disrupt homeostasis of the prey through several mechanisms, including among others, blockage of cyclic nucleotide-gated and voltage-gated ion channels and inhibition of smooth muscle contraction. We evaluated the molecular evolution of CRISPs in toxicoferan reptiles at both nucleotide and protein levels relative to their nonvenomous mammalian homologs. We show that the evolution of CRISP gene in these reptiles is significantly influenced by positive selection and in snakes (ω = 3.84) more than in lizards (ω = 2.33), whereas mammalian CRISPs were under strong negative selection (CRISP1 = 0.55, CRISP2 = 0.40, and CRISP3 = 0.68). The use of ancestral sequence reconstruction, mapping of mutations on the three-dimensional structure, and detailed evaluation of selection pressures suggests that the toxicoferan CRISPs underwent accelerated evolution aided by strong positive selection and directional mutagenesis, whereas their mammalian homologs are constrained by negative selection. Gene and protein-level selection analyses identified 41 positively selected sites in snakes and 14 sites in lizards. Most of these sites are located on the molecular surface (nearly 76% in snakes and 79% in lizards), whereas the backbone of the protein retains a highly conserved structural scaffold. Nearly 46% of the positively selected sites occur in the cysteine-rich domain of the protein. This directional mutagenesis, where the hotspots of mutations are found on the molecular surface and functional domains of the protein, acts as a diversifying mechanism for the exquisite biological targeting of CRISPs in toxicoferan reptiles. Finally, our analyses suggest that the evolution of toxicoferan-CRISP venoms might have been influenced by the specific predatory mechanism employed by the organism. CRISPs in Elapidae, which mostly employ neurotoxins, have experienced less positive selection pressure (ω = 2.86) compared with the "nonvenomous" colubrids (ω = 4.10) that rely on grip and constriction to capture the prey, and the Viperidae, a lineage that mostly employs haemotoxins (ω = 4.19). Relatively lower omega estimates in Anguimorph lizards (ω = 2.33) than snakes (ω = 3.84) suggests that lizards probably depend more on pace and powerful jaws for predation than venom.     

Plant morphophysiological and anatomical factors associated with nitrous oxide flux from wheat (Triticum aestivum)

Experiments were conducted to study the dynamics of nitrous oxide (N?O) emission from wheat varieties viz., Sonalika, HUW 468, HUW 234 and DBW 14 grown in alluvial soils of North Bank Plain Agroclimatic Zone of Assam, India. Attempts were made to find out the relationship of N?O emission with plant morphophysiological, anatomical and soil properties. N?O fluxes from wheat varieties ranged from 40 μg N?O-N m?2 h?1 to 295 μg N?O-N m?2 h?1. Soil organic carbon and soil temperature have shown significant relationship with N?O flux. The rate of leaf transpiration recorded from the wheat varieties at different growth stages exhibited a positive correlation with N?O emission suggesting that movement of N?O along with the transpirational water flow may be an important mechanism of N?O transport and emission through wheat plants. Anatomical investigation by scanning electron microscope revealed that N?O emission has relationship with stomatal frequency of leaf and leaf sheaths. Variety HUW 234 with the highest stomatal frequency of leaf and leaf sheath also recorded higher seasonal N?O emission compared to other varieties. Seasonal N?O emission (E(sif)) of the varieties ranged from 3.25 to 3.81 kg N?O-N ha?1. Significant variations in E(sif) values were recorded within the varieties.     

A panel of microsatellites to individually identify leopards and its application to leopard monitoring in human dominated landscapes

Background

Copy number variants (CNVs) have been identified in several studies to be associated with complex diseases. It is important, therefore, to understand the distribution of CNVs within and among populations. This study is the first report of a CNV map in African Americans.

Results

Employing a SNP platform with greater than 500,000 SNPs, a first-generation CNV map of the African American genome was generated using DNA from 385 healthy African American individuals, and compared to a sample of 435 healthy White individuals. A total of 1362 CNVs were identified within African Americans, which included two CNV regions that were significantly different in frequency between African Americans and Whites (17q21 and 15q11). In addition, a duplication was identified in 74% of DNAs derived from cell lines that was not present in any of the whole blood derived DNAs.

Conclusion

The Affymetrix 500 K array provides reliable CNV mapping information. However, using cell lines as a source of DNA may introduce artifacts. The duplication identified in high frequency in Whites and low frequency in African Americans on chromosome 17q21 reflects haplotype specific frequency differences between ancestral groups. The generation of the CNV map will be a valuable tool for identifying disease associated CNVs in African Americans.     

Secretory immunoglobulin A antibodies against the sigma1 outer capsid protein of reovirus type 1 Lang prevent infection of mouse Peyer's patches

Reovirus type 1 Lang (T1L) adheres to M cells in the follicle-associated epithelium of mouse intestine and exploits the transport activity of M cells to enter and infect the Peyer's patch mucosa. Adult mice that have previously cleared a reovirus T1L infection have virus-specific immunoglobulin G (IgG) in serum and IgA in secretions and are protected against reinfection. Our aim in this study was to determine whether secretory IgA is sufficient for protection of Peyer's patches against oral reovirus challenge and, if so, against which reovirus antigen(s) the IgA may be directed. Monoclonal antibodies (MAbs) of the IgA isotype, directed against the sigma1 protein of reovirus T1L, the viral adhesin, were produced and tested along with other, existing IgA and IgG MAbs against reovirus T1L outer capsid proteins. Anti-sigma1 IgA and IgG MAbs neutralized reovirus T1L in L cell plaque reduction assays and inhibited T1L adherence to L cells and Caco-2(BBe) intestinal epithelial cells in vitro, but MAbs against other proteins did not. Passive oral administration of anti-sigma1 IgA and IgG MAbs prevented Peyer's patch infection in adult mice, but other MAbs did not. When anti-sigma1 IgA and IgG MAbs were produced in mice from hybridoma backpack tumors, however, the IgA prevented Peyer's patch infection, but the IgG did not. The results provide evidence that neutralizing IgA antibodies specific for the sigma1 protein are protective in vitro and in vivo and that the presence of these antibodies in intestinal secretions is sufficient for protection against entry of reovirus T1L into Peyer's patches.     

Structure of the N-terminal half of gelsolin bound to actin: roles in severing, apoptosis and FAF

The actin filament-severing functionality of gelsolin resides in its N-terminal three domains (G1-G3). We have determined the structure of this fragment in complex with an actin monomer. The structure reveals the dramatic domain rearrangements that activate G1-G3, which include the replacement of interdomain interactions observed in the inactive, calcium-free protein by new contacts to actin, and by a novel G2-G3 interface. Together, these conformational changes are critical for actin filament severing, and we suggest that their absence leads to the disease Finnish-type familial amyloidosis. Furthermore, we propose that association with actin drives the calcium-independent activation of isolated G1-G3 during apoptosis, and that a similar mechanism operates to activate native gelsolin at micromolar levels of calcium. This is the first structure of a filament-binding protein bound to actin and it sets stringent, high-resolution limitations on the arrangement of actin protomers within the filament.     

Biophysical characterization and molecular modeling of the coordinative-intercalative DNA monoadduct of a platinum-acridinylthiourea agent in a site-specifically modified dodecamer

The guanine-N7 monoadduct of [Pt(en)Cl(ACRAMTU)](NO₃)₂ (PT-ACRAMTU; en=ethane-1,2-diamine, ACRAMTU=1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea), a dual metalating/intercalating cytotoxic agent, was generated in a double-stranded dodecamer, d(CCTCTCG*TCTCC/GGAGACGAGAGG) (III*), and isolated by preparative reverse-phase high-performance liquid chromatography (HPLC). The adduct was characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), circular-dichroism spectropolarimetry (CD), UV-melting curves, and NMR spectroscopy. In addition, a molecular mechanics/restrained molecular dynamics (MM/rMD) study was performed for this adduct using the AMBER force field. Monoadduction of the sequence leads to a pronounced increase in melting temperature, T_m=T_m(III*)–T_m(III)=9.7 °C. Because there is complete enthalpy–entropy compensation, binding occurs without noticeable thermodynamic destabilization. This feature and the CD (induced-ligand circular dichroism) and NMR (upfield shifts of aromatic acridine proton signals) data are indicative of a unique, nondenaturing dual-binding mode that involves partial intercalation of the acridine chromophore. An energy-minimized AMBER model of III* demonstrates that platination of G7-N7 of guanine in the major groove and partial insertion of the acridine moiety into the C6G19/G7C18 base step on the 5 face of the modified purine base is feasible and supportive of the experimental results. Differences in the biophysical properties between III* and duplexes containing adducts of the clinical-drug cisplatin are outlined, and possible biological consequences are discussed.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0534-3Abbreviations ACRAMTU 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea - dGuo 2-deoxyguanosine - dGuo* [Pt(en)(ACRAMTU-S)(dGuo-N7)]³⁺ - en ethane-1,2-diamine - ICD Induced circular dichroism - MALDI-TOF MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry - MM Molecular mechanics - PIPES 1,4-piperazinediethanesulfonic acid - PT-ACRAMTU [Pt(en)Cl(ACRAMTU)](NO₃)₂ - rMD Restrained molecular dynamics