Anticonvulsant sugar sulfamates. Potent cyclic sulfate and cyclic sulfite analogues of topiramate

Exploration structure-activity relationships surrounding the clinically effective antiepileptic drug topiramate (1) led to a series of potent anticonvulsants with a 4,5-cyclic sulfate or 4,5-cycli sulfite functionality. Key derivative 2 (RWJ-37947) is ca. 8 times more potent than topiramate in mice; it also features a long duration of action and a very favorable neurotoxicity index.     

The agony of choice: Impact of the host animal species on the enzyme-linked immunosorbent assay performance for host cell protein quantification

Host cell proteins (HCPs) are inevitable process-related impurities in biotherapeutics commonly monitored by enzyme-linked immunosorbent assays (ELISAs). Of particular importance for their reliable detection are the anti-HCP polyclonal antibodies (pAbs), supposed to detect a broad range of HCPs. The present study focuses on the identification of suitable host animal species for the development of high-performance CHO-HCP ELISAs, assuming the generation of pAbs with adequate coverage and specificity. Hence, antibodies derived from immunization of sheep, goats, donkeys, rabbits, and chickens were compared concerning their amount of HCP-specific antibodies, coverage, and performance in a sandwich ELISA. Immunization of sheep, goats, donkeys, and rabbits met all test criteria, whereas the antibodies from chickens cannot be recommended based on the results of this study. Additionally, a mixture of antibodies from the five host species was prepared to assess if coverage and ELISA performance can be improved by a multispecies approach. Comparable results were obtained for the single- and multispecies ELISAs in different in-process samples, indicating no substantial improvement for the latter in ELISA performance while raising ethical and financial concerns.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Recovery of the coral Montastrea annularis in the Florida Keys after the 1987 Caribbean “bleaching event”

Many reef-building corals and other cnidarians lost photosynthetic pigments and symbiotic algae (zooxanthellae) during the coral bleaching event in the Caribbean in 1987. The Florida Reef Tract included some of the first documented cases, with widespread bleaching of the massive coral Montastrea annularis beginning in late August. Phototransects at Carysfort Reef showed discoloration of >90% of colonies of this species in March 1988 compared to 0% in July 1986; however no mortality was observed between 1986 and 1988. Samples of corals collected in February and June 1988 had zooxanthellae densities ranging from 0.1 in the most lightly colored corals, to 1.6x10⁶ cells/cm² in the darker corals. Minimum densities increased to 0.5x10⁶ cells/cm² by August 1989. Chlorophyll-a content of zooxanthellae and zooxanthellar mitotic indices were significantly higher in corals with lower densities of zooxanthellae, suggesting that zooxanthellar at low densities may be more nutrientsufficient than those in unbleached corals. Ash-free dry weight of coral tissue was positively correlated with zooxanthellae density at all sample times and was significantly lower in June 1988 compared to August 1989. Proteins and lipids per cm² were significantly higher in August 1989 than in February or June, 1988. Although recovery of zooxanthellae density and coral pigmentation to normal levels may occur in less than one year, regrowth of tissue biomass and energy stores lost during the period of low symbiont densities may take significantly longer.     

Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

βVI Turns in peptides and proteins: A model peptide mimicry

To investigate the role of proline in defining β turn conformations within cyclic hexa- and pentapeptides we synthesized and determined the conformations of a series of L - and D -proline-containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L -proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D -proline peptides. The cis conformations of the compounds cyclo(-Pro-Ala-Ala-Pro-Ala-Ala-), cyclo(-Arg-Gly-Asp-Phe-Pro-Gly-), cyclo(-Arg-Gly-Asp-Phe-Pro-Ala-), cyclo(-Pro-Ala-Ala-Ala-Ala--), and cyclo(-Pro-Ala-Pro-Ala-Ala-) form uncommon βVI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline-following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg-Gly-Asp), are incorporated. © 1993 Wiley-Liss, Inc.     

Correlation between protein stability and crystal properties of designed ROP variants

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.     

Hidden diversity in high-latitude Southern Hemisphere environments: Reinstatement of the genus Rama and description of Vandenhoekia gen. nov. (Cladophoraceae,Ulvophyceae, Chlorophyta), two highly variable genera

The continental coasts and remote islands in the high-latitude Southern Hemisphere, including the subantarctic region, are characterized by many endemic species, high abundance of taxa, and intermediate levels of biodiversity. The macroalgal flora of these locations has received relatively little attention. Filamentous green algae are prolific in the intertidal of southern islands, but the taxonomy, distribution, and evolutionary history of these taxa are yet to be fully explored, mostly due to the difficulty of access to some of these locations. In this study, we examined specimens of the order Cladophorales from various locations in the high-latitude Southern Hemisphere including the subantarctic (the Auckland Islands, Bounty Islands, Campbell Island, Macquarie Island, and Kerguelen Islands), as well as mainland New Zealand, the Chatham Islands, Chile, and Tasmania. The analyses of the rDNA sequences of the samples revealed the existence of two new clades in a phylogeny of the Cladophoraceae. One of these clades is described as the novel genus Vandenhoekia gen. nov., which contains three species that are branched or unbranched. The amended genus Rama is reinstated to accommodate the other clade, and contains four species, including the Northern Hemisphere “Cladophora rupestris.” In Rama both branched and unbranched morphologies are found. It is remarkable that gross morphology is not a predictor for generic affiliations in these algae. This study illustrates that much can still be learned about diversity in the Cladophorales and highlights the importance of new collections, especially in novel locations.     

Chromosomal mapping in the mouse of eight K-channel-genes representing the four Shaker-like subfamilies Shaker, Shab, Shaw, and Shal

The four Shaker-like subfamilies of Shaker-, Shab-,Shaw-, and Shal-related K⁺ channels in mammals have been defined on the basis of their sequence homologies to the corresponding Drosophila genes. Using interspecific backcrosses between Mus musculus and Mus spretus, we have chromosomally mapped in the mouse the Shaker-related K⁺-channel genes Kcna1, Kcna2, Kcna4, Kcna5, and Kcna6; the Shab-related gene Kcnb1; the Shaw-related gene Kcnc4; and the Shal-related gene Kcnd2. The following localizations were determined: Chr 2, cen-Acra-Kcna4-Pax-6-a-Pck-1-Kras-3-Kcnb1 (corresponding human Chrs 11p and 20q, respectively); Chr 3, cen-Hao-2-(Kcna2, Kcnc4)-Amy-1 (human Chr 1); and Chr 6, cen-Cola-2-Met-Kcnd2-Cpa-Tcrb-adr/Clc-1-Hox-1.1-Myk-103-Raf-1-(Tpi-1, Kcna1, Kcna5, Kcna6) (human Chrs 7q and 12p, respectively). Thus, there is a cluster of at least three Shaker-related K⁺-channel genes on distal mouse Chr 6 and a cluster on Chr 2 that at least consists of one Shaker-related and one Shaw-related gene. The three other K⁺-channel genes are not linked to each other. The map positions of the different types of K⁺-channel genes in the mouse are discussed in relation to those of their homologs in man and to hereditary diseases of mouse and man that might involve K⁺ channels.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.