Biological and cytoselective anticancer properties of copper(II)-polypyridyl complexes modulated by auxiliary methylated glycine ligand

A series of ternary copper(II)-1,10-phenanthroline complexes with glycine and methylated glycine derivatives, [Cu(phen)(aa)(H(2)O)]NO(3)·xH(2)O 1-4 (amino acid (aa): glycine (gly), 1; DL: -alanine (DL: -ala), 2; 2,2-dimethylglycine (C-dmg), 3; sarcosine (sar), 4), were synthesized and characterized by FTIR, elemental analysis, electrospray ionization-mass spectra (ESI-MS), UV-visible spectroscopy and molar conductivity measurement. The determined X-ray crystallographic structures of 2 and 3 show each to consist of distorted square pyramidal [Cu(phen)(aa)(H(2)O)](+) cation, a nitrate counter anion, and with or without lattice water, similar to previously reported structure of [Cu(phen)(gly)(H(2)O)]NO(3)·1?H(2)O. It is found that 1-4 exist as 1:1 electrolytes in aqueous solution, and the cationic copper(II) complexes are at least stable up to 24?h. Positive-ion ESI-MS spectra show existence of only undissociated [Cu(phen)(aa)](+) species. Electron paramagnetic resonance, gel electrophoresis, fluorescence quenching, and restriction enzyme inhibition assay were used to study the binding interaction, binding affinity and selectivity of these complexes for various types of B-form DNA duplexes and G-quadruplex. All complexes can bind selectively to DNA by intercalation and electrostatic forces, and inhibit topoisomerase I. The effect of the methyl substituents of the coordinated amino acid in the above complexes on these biological properties are presented and discussed. The IC(50) values (24?h) of 1-4 for nasopharyngeal cancer cell line HK1 are in the range 2.2-5.2?μM while the corresponding values for normal cell line NP69 are greater than 13.0?μM. All complexes, at 5?μM, induced 41-60?% apoptotic cell death in HK1 cells but no significant cell death in NP69 cells.     

Cloning and characterisation of the Schizosaccharomyces pombe rad8 gene, a member of the SNF2 helicase family.

The Schizosaccharomyces pombe rad8 mutant is sensitive to both UV and gamma irradiation. We have cloned the rad8 gene by complementation of the UV sensitivity of a rad8.190 mutant strain. The gene comprises an open reading frame of 3.4 kb which does not contain any introns and is capable of encoding a 1133 amino acid protein of 129 kDa. Deletion of the gene indicates that it is not essential for cell viability. Recognisable motifs are present for a nuclear localisation signal, a RING finger and helicase domains. The predicted protein is a member of the SNF2 subfamily of proteins and shows particular homology to the Saccharomyces cerevisiae RAD5 protein. Double mutant analysis demonstrated that the rad8 mutant is not epistatic to mutants in the excision repair pathway (rad13) or checkpoint pathway (rad9). Analysis of radiation sensitivity though the cell cycle indicates that, unlike most other rad mutants, rad8 is most sensitive to irradiation during the G1/S period.     

Strategies for selecting Recombinant CHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors

Manufacture of recombinant proteins from mammalian cell lines requires the use of bioreactor systems at scales of up to 20,000 L. The cost and complexity of such systems can prohibit their extensive use during the process to construct and select the manufacturing cell line. It is therefore common practice to develop a model of the production process in a small scale vessel, such as a shake‐flask, where lower costs, ease of handling, and higher throughput are possible. This model can then be used to select a small number of cell lines for further evaluation in bioreactor culture. Here, we extend our previous work investigating cell line construction strategies to assess how well the behavior of cell lines in such a shake‐flask assessment predicts behavior in the associated bioreactor production process. A panel of 29 GS‐CHO cell lines, all producing the same antibody, were selected to include a mixture of high and low producers from a pool of 175 transfectants. Assessment of this panel in 10 L bioreactor culture revealed wide variation in parameters including growth, productivity, and metabolite utilization. In general, those cell lines which were high producing in the bioreactor cultures had also been higher producing in an earlier shake‐flask assessment. However, some changes in rank position of the evaluated cell lines were seen between the two systems. A potential explanation of these observations is discussed and approaches to improve the predictability of assessments used for cell line selection are considered. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.     

Veterinary medicine in the 21st century: the challenge of biosecurity

The veterinary profession is presently challenged with developing and maintaining on-farm biosecurity protocols to protect the nation's food supply from acts of bioterrorism, from the growing threat of foreign animal diseases, and from multidrug resistance among pathogenic organisms. This challenge comes at a time when the supply of food animal veterinarians in the United States is progressively in decline, and raises the possibility that the profession is not adequately prepared to fulfill its responsibilities to the health and productivity of the US livestock and poultry populations. Causes of the decline in demand for veterinary services are discussed. They include consolidation of the food animal industries and a trend toward transferring performance of tasks traditionally carried out by veterinarians to the province of lay staff. This development potentially reduces veterinary surveillance of food animal populations. It also runs the risk of delay in recognizing and controlling serious health problems when they arise. Several remedies are proposed, including profound changes in the curriculum for educating food animal veterinarians to serve the consolidated but vulnerable livestock and poultry industries suitably. Also advocated is the initiation of training programs for herdsmen on the symptoms of foreign animal diseases, together with advice on when to call a veterinarian. Significant investment of federal or state resources will be required if these changes are to become reality.     

Ii-Key/HER-2/<Emphasis Type="Italic">neu</Emphasis> MHC class-II antigenic epitope vaccine peptide for breast cancer

Purpose Cytotoxic T lymphocytes (CTL)- and T-helper cell-specific, and major histocompatibility complex (MHC) class-I and class-II peptides, respectively, of the HER-2/neu protein, induce immune responses in patients. A major challenge in developing cancer peptide vaccines is breaking tolerance to tumor-associated antigens which are functionally self-proteins. An adequate CD4+ T-helper response is required for effective and lasting responses.Methods Stimulating anti-cancer CD4+ T cell responses by MHC class-II epitope peptides has been limited by their weak potency, at least compared with tight-binding MHC class-I epitope peptides. Previously, a potent T-cell response to a MHC class-II epitope was engineered by coupling the N-terminus of the pigeon cytochrome C [PGCC(95–104)] MHC class-II epitope to the C-terminus of an immunoregulatory segment of the Ii protein (hIi77–81, the Ii-Key peptide) through a polymethylene spacer.Results In vitro presentation of the MHC class-II epitope to a T hybridoma was enhanced greatly (>250 times). Now, an Ii-Key/HER-2/neu (777–789) MHC class-II epitope hybrid peptide stimulated lymphocytes from both a healthy donor and a patient with metastatic breast carcinoma. The in vitro primary stimulation with the hybrid peptide strongly activated IFN- release, whereas the epitope-only peptide was weakly active. In fact, the hybrid stimulated IFN- release as well as the wild-type peptide when augmented with IL-12; however, the hybrid was comparable to free peptide in stimulating IL-4 release. This pattern is consistent with preferential activation along a non-tolerogenic Th1 pathway.Conclusion Such Ii-Key/MHC class-II epitope hybrid peptides have both diagnostic and therapeutic applications.     

Expression and function of T cell homing molecules in Hodgkin’s lymphoma

Circulating T lymphocytes enter a tissue if they express appropriate chemokine receptors and adhesion molecules to engage ligands presented at this site. To aid rational development of T cell-based therapies for Hodgkin's lymphoma (HL), we have assessed the expression and function of homing receptors on tumour-infiltrating T cells in HL and compared them with T cells from unaffected lymph nodes and colorectal cancer tissue. Chemokine receptors CXCR3, CXCR4 and CCR7 were expressed on a large proportion of T cells within HL tissue and mediated chemotaxis to purified chemokine. The corresponding ligands (CXCL10, CXCL12, CCL21) were expressed on the malignant cells and/or vascular endothelium. Adhesion molecules including CD62L were widely expressed on HL-derived T cells and their corresponding ligands were detected on vessels within the tumour. This homing phenotype was distinct from T cells isolated from colorectal cancer, but matched closely the phenotype of T cells from unaffected lymph nodes. Thus, T cell recruitment to HL resembles entry of na?ve/central memory T cells into normal lymph nodes. This has important implications for current approaches to treat HL using T cells activated and expanded in vitro that lack CCR7 and CD62L expression.     

Amplified Fragment Length Polymorphism Analysis of Campylobacter jejuni Strains Isolated from Chickens and from Patients with Gastroenteritis or Guillain-Barré or Miller Fisher Syndrome

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between Campylobacter jejuni strains infecting chickens (n = 54) and those causing gastroenteritis in humans (n = 53). In addition, C. jejuni strains associated with the development of Guillain-Barré syndrome (GBS) (n = 14) and Miller Fisher syndrome (MFS) (n = 4), two related acute paralytic syndromes in human, were included. Strains were isolated between 1989 and 1998 in The Netherlands. The AFLP banding patterns were analyzed with correlation-based and band-based similarity coefficients and UPGMA (unweighted pair group method using average linkages) cluster analysis. All C. jejuni strains showed highly heterogeneous fingerprints, and no fingerprints exclusive for chicken strains or for human strains were obtained. All strains were separated in two distinct genetic groups. In group A the percentage of human strains was significantly higher and may be an indication that genotypes of this group are more frequently associated with human diseases. We conclude that C. jejuni from chickens cannot be distinguished from human strains and that GBS or MFS related strains do not belong to a distinct genetic group.     

A new species of Cytauxzoon from Pallas' cats caught in Mongolia and comments on the systematics and taxonomy of piroplasmids

DNA was extracted and the 18S ribosomal RNA gene was amplified and sequenced from the blood of 2 Pallas' cats (Otocolobus manul) infected with small intraerythrocytic piroplasms. Sequences of the parasite were found to be identical with that of a previously reported Cytauxzoon-like piroplasm from a Pallas' cat. Phylogenetic analyses of the parasite DNA sequences obtained from the 3 Pallas' cats to other piroplasms revealed a sister group relationship to C. felis. The mean corrected percent sequence divergence between the Pallas' cat parasite and C. felis was 1.490%, which is greater than that for most other piroplasms in which species status has been accepted. On the basis of the sequence variation, we propose to name the Pallas' cat parasite C. manul. Phylogenetic analyses of C. manul also revealed a close relationship with the Spanish Cytauxzoon-like isolate because they exhibited only 0.389% sequence divergence, yet these sequences exhibit a mean of 1.690% sequence divergence from the New World isolate of C. felis. Our phylogenetic analyses also revealed several taxonomic problems that have impeded the development of a classification that accurately reflects evolutionary history of piroplasms. As currently arranged, Babesia and Theileria are paraphyletic taxa and are in need of reorganization.