Comparison of redox and ligand binding behaviour of yeast and bovine cytochrome c oxidases using FTIR spectroscopy

Redox and CO photolysis FTIR spectra of yeast cytochrome c oxidase WT and mutants are compared to those from bovine and P. denitrificans CcOs in order to establish common functional features. All display changes that can be assigned to their E242 (bovine numbering) equivalent and to weakly H-bonded water molecules. The additional redox-sensitive band reported at 1736?cm^?1 in bovine CcO and previously assigned to D51 is absent from yeast CcO and couldn't be restored by introduction of a D residue at the equivalent position of the yeast protein. Redox spectra of yeast CcO also show much smaller changes in the amide I region, which may relate to structural differences in the region around D51 and the subunit I/II interface.     

Conformational analysis of cyclic angiotensin II analogues

Summary Conformationally restricted cyclic analogues of angiotensin II (ANG II), Asp¹-Arg²-Val³-Tyr⁴-Val⁵-His⁶-Pro⁷-Phe⁸, with a link between positions 3 and 5 have considerable biological activity. It is proposed that the spatial arrangement of the pharmacophore groups of Tyr⁴, His⁶ and Phe⁸ side chains and the C-terminal carboxyl group in ANG II and active analogues is similar. Conformational analysis of ANG II and two cyclic analogues c[Sar¹, Lys³,Glu⁵]ANG II and c[Sar¹,Hcy³,Mpt⁵]ANG II was performed, and a geometrical comparison of the low-energy conformations of these compounds allowed one to propose a model of receptor-bound conformation in terms of the spatial arrangement of the pharmacophore groups. This model is characterised by the close spatial location of the His⁶-Phe⁸ side chains and the Tyr⁴ C-terminal carboxyl group and is stabilised by the electrostatic interaction of Arg² and the C-terminal carboxyl group.Abbreviations ANG II angiotensin II - Hcy homocysteine - Mpt trans-4-mercaptoproline     

N-glycan structures of a recombinant mouse soluble Fcγ receptor II

N-glycans of a recombinant mouse soluble Fc receptor II (sFcRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was -(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and -(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.     

Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase II alpha

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.     

Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorometer

A newly developed modulation fluorometer is described which operates with 1 sec light pulses from a light-emitting diode (LED) at 100 KHz. Special amplification circuits assure a highly selective recording of pulse fluorescence signals against a vast background of non-modulated light. The system tolerates ratios of up to 1:10⁷ between measuring light and actinic light. Thus it is possible to measure the dark fluorescence yield and record the kinetics of light-induced changes. A high time resolution allows the recording of the rapid relaxation kinetic following a saturating single turnover flash. Examples of system performance are given. It is shown that following a flash the reoxidation kinetics of photosystem II acceptors are slowed down not only by the inhibitor DCMU, but by a number of other treatments as well. From a light intensity dependency of the induction kinetics the existence of two saturated intermediate levels (I₁ and I₂) is apparent, which indicates the removal of three distinct types of fluorescence quenching in the overall fluorescence rise from F₀ to F_max.Abbreviations Q_A and Q_B consecutive electron acceptors of photosystem II - PS II photosystem II - P 680 reaction center chlorophyll of photosystem II - F₀ minimum fluorescence yield following dark adaptation - F_max maximum fluorescence yield - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea - DCCD N,N-dicyclohexylcarbodiimide - PQ plastoquinone - DAD diaminodurene Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

The effect of the polyproline II (PPII) conformation on the denatured state entropy

Polyproline II (PPII) is reported to be a dominant conformation in the unfolded state of peptides, even when no prolines are present in the sequence. Here we use isothermal titration calorimetry (ITC) to investigate the PPII bias in the unfolded state by studying the binding of the SH3 domain of SEM-5 to variants of its putative PPII peptide ligand, Sos. The experimental system is unique in that it provides direct access to the conformational entropy change of the substituted amino acids. Results indicate that the denatured ensemble can be characterized by at least two thermodynamically distinct states, the PPII conformation and an unfolded state conforming to the previously held idea of the denatured state as a random collection of conformations determined largely by hard-sphere collision. The probability of the PPII conformation in the denatured states for Ala and Gly were found to be significant, approximately 30% and approximately 10%, respectively, resulting in a dramatic reduction in the conformational entropy of folding.     

Synthesis and anti-cancer activity of bis-amino-phosphine ligand and its ruthenium(II) complexes

The development of both chemotherapeutic drug resistance as well as adverse side effects suggest that the current chemotherapeutic drugs remain ineffective in treating the various types of cancers. The development of new metallodrugs presenting anti-cancer activity is therefore needed. Ruthenium complexes have gained a great deal of interest due to their promising anti-tumour properties and reduced toxicity in vivo. This study highlighted the effective induction of cell death in a malignant melanoma cell by two novel bis-amino-phosphine ruthenium(II) complexes referred to as GA105 and GA113. The IC₅₀ concentrations were determined for both the complexes, the ligand and cisplatin, for comparison. Both complexes GA105 and GA113 displayed a high anti-cancer selectivity profile as they exhibited low IC₅₀ values of 6.72 µM and 8.76 µM respectively, with low toxicity towards a non-malignant human cell line. The IC₅₀ values obtained for both complexes were lower than that of cisplatin. The new complexes were more effective compared to the free ligand, GA103 (IC₅₀ = >20 µM). Morphological studies on treated cells induced apoptotic features, which with further studies could indicate an intrinsic cell death pathway. Additionally, flow cytometric analysis revealed that the mode of cell death of complex GA113 was apoptosis. The outcomes herein give further insight into the potential use of selected Ru(II) complexes as alternative chemotherapeutic drugs in the future.     

A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane‐specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CAR riers of the T GN to the cell S urface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)‐G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.