Novel inhibitors of plasminogen activator inhibitor-1: development of new templates from diketopiperazines

Several isoquinoline-based templates were identified from the studies of the conformational effects of the diketopiperazine structures for PAI-1 inhibition. Moderate to good activity was retained with the elimination of unattractive characteristics in the diketopiperazine template.     

Structure of ferricytochrome c' from Rhodospirillum molischianum at 1.67 A resolution

The structure of ferricytochrome c' from Rhodospirillum molischianum has been crystallographically refined to 1.67 A resolution using a combination of reciprocal space and restrained least-squares refinement methods. The final crystallographic R-factor for 30,533 reflections measured with I greater than sigma (I) between infinity and 1.67 A is 0.188. The final model incorporates 1944 unique protein atoms (of a total of 1972) together with 194 bound solvent molecules. The structure has been analysed with respect to its detailed conformational properties, secondary structural features, temperature factor behavior, bound solvent sites, and heme geometry. The asymmetric unit of the cytochrome c' crystal contains a dimer composed of chemically identical 128-residue polypeptide chains. Although the refined structure shows the monomers to be very similar, examination of the differences that do occur allows an evaluation of how different lattice contacts affect protein conformation and solvent binding. In particular, comparison of solvent binding sites in the two subunits allows identification of a common set that are not altered by lattice interactions. The preservation of these solvent interactions in different lattice environments suggests that they play a structural role in protein stabilization in solution. The refined structure additionally reveals some new features that relate to the ligand binding properties and unusual mixed-spin state character of cytochrome c'. Finally, comparison of the heme binding geometry in cytochrome c' and other structurally unrelated c-type cytochromes shows that two alternative, but sterically favorable, conformational variants occur among the seven examples examined.     

Construction and Characterization of External Cavity Diode Lasers for Atomic Physics

Since their development in the late 1980s, cheap, reliable external cavity diode lasers (ECDLs) have replaced complex and expensive traditional dye and Titanium Sapphire lasers as the workhorse laser of atomic physics labs^1,2. Their versatility and prolific use throughout atomic physics in applications such as absorption spectroscopy and laser cooling^1,2 makes it imperative for incoming students to gain a firm practical understanding of these lasers. This publication builds upon the seminal work by Wieman³, updating components, and providing a video tutorial. The setup, frequency locking and performance characterization of an ECDL will be described. Discussion of component selection and proper mounting of both diodes and gratings, the factors affecting mode selection within the cavity, proper alignment for optimal external feedback, optics setup for coarse and fine frequency sensitive measurements, a brief overview of laser locking techniques, and laser linewidth measurements are included.     

Systematic relationships of <Emphasis Type=Italic>Mosgovoyia</Emphasis> Spasskii, 1951 (Cestoda: Anoplocephalidae) and related genera inferred from mitochondrial and nuclear sequence data

The present study evaluates the phylogenetic position and systematic relationships of two species of Mosgovoyia Spasskii, 1951 and related genera (Cestoda: Anoplocephalidae) based on sequences of 28S ribosomal RNA and mitochondrial NADH dehydrogenase subunit 1 (Nad1) genes. Both molecular data-sets show that M. pectinata (Goeze, 1782) and Schizorchis caballeroi Rausch, 1960 are sister species and that they are phylogenetically independent from M. ctenoides (Railliet, 1890). This shows unambiguously that Mosgovoyia [sensu Beveridge (1978)] is a non-monophyletic assemblage, supporting the validity of Neoctenotaenia Tenora, 1976, erected for M. ctenoides. The results also show that the morphologically related Ctenotaenia marmotae (Fröhlich, 1802) is the sister species of Andrya rhopalocephala (Riehm, 1881) and therefore represents a more derived lineage. Modified diagnoses are provided for Mosgovoyia and Neoctenotaenia.     

Morphological and molecular characterisation of Paranoplocephala buryatiensis n. sp. and P. longivaginata Chechulin & Gulyaev, 1998 (Cestoda: Anoplocephalidae) in voles of the genus Clethrionomys

A new species, Paranoplocephala buryatiensis n. sp. (Cestoda:Anoplocephalidae), is described from the grey-sided vole Clethrionomys rufocanus (Sundevall) in the Republic of Buryatia (Russian Federation) and compared with P. longivaginata Chechulin & Gulyaev, 1998, a parasite of the red vole C. rutilus (Pallas) in the same region. P. buryatiensis n. sp. and P. longivaginata both have an exceptionally long vagina and cirrus, unique features among known species of Paranoplocephala Lühe, 1910. The new species differs from P. longivaginata primarily by its wider and more robust body, lower length/width ratio of mature proglottides, tendency of testes to occur in two separate groups, seminal receptacle of a different shape and the position of the cirrus-sac with respect to the ventral longitudinal osmoregulatory canal. The cytochrome oxidase subunit I (COI) sequence data support the independent status of these species, and show that they form a monophyletic assemblage within Paranoplocephala (sensu lato). Assuming cospeciation, an indirect calibration using host speciation dates estimated a rate of mtDNA substitution of 1.0–1.7% pairwise (0.5–0.85% per lineage) sequence divergence per million years. A faunistic review of Paranoplocephala species in C. rufocanus and C. rutilus in the Holarctic region is presented.     

Efficient photosensitization of malignant human cells in vitro by liposome-bound porphyrins

Comparative kinetics of porphyrin uptake and release by HeLa cells, incubated with equivalent concentrations of either hematoporphyrin (Hp) in aqueous solution or Hp and its dimethylester (HpDME) bound to unilamellar liposomes, show that liposomal porphyrins are bound at a higher rate and in considerably larger amounts. Moreover, the release of cell-bound porphyrins into the medium is remarkably reduced and slowered after cell loading with liposome-bound porphyrins. The presence of 1% bovine or human serum albumin (but not serum globulins) in the medium has no effect on uptake and release of liposome-bound porphyrins by HeLa cells, whereas it remarkably decreases the uptake of aqueous Hp. Parallel studies of cell photodamages under known concentrations of cell-bound porphyrin unequivocally demonstrate that the photodynamic effect is strictly related to the porphyrin load. As a consequence a dramatic increase of cell-photosensitizing efficiency is obtained by binding Hp (and even more HpDME) with liposomes. Electron microscopy investigations on cell damages caused by loading with liposome-bound porphyrins and subsequent illumination show that the plasmatic membrane is one important cell site of porphyrin interaction and photodynamic effect.     

Sterols of Testudodinium testudo (formerly Amphidinium testudo): Production of the Δ8(14) sterol gymnodinosterol and chemotaxonomic relationship to the Kareniaceae

Testudodinium testudo is a peridinin-containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ⁸⁽¹⁴⁾ sterols, such as 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ⁸⁽¹⁴⁾ sterol, (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α-methyl-5α-ergosta-8,14,22-trien-3β-ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.     

Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody

Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.