A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC₅₀<10 nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5.     

The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection

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New liverwort records for Vietnam

A short collecting expedition to Vietnam in 2016 yielded 700 specimens of 170 liverwort species of which 79 were new to the flora of Vietnam, including 43 species new to Indochina. Many of the newly recorded species have meta-Himalayan distributions (that predicts occurrence of these taxa in the areas of north-west Vietnam situated in the south-easternmost spurs of the Himalaya). Some species were recorded based on the study of living material, permitting the examination of oil body characteristics for Riccardia and Calypogeia. The known taxonomic liverwort diversity of Vietnam is currently 379 taxa, estimated to represent ca 80% of the actual taxonomic diversity in the country.     

Isolation and long-term culture of gallbladder epithelial cells from wild-type and CF mice

Summary Mice with targeted disruption of the cftr gene show pathophysiologic changes in the gallbladder, which correlate with hepatobiliary disease seen in cystic fibrosis patients. As gallbladder epithelium secretes mucin, and as this epithelium consists of a relatively homogenous cell type, study of CFTR function in these cells would be beneficial to delineate the complex cellular functions of this protein. The size and anatomic location of the murine gallbladder makes such studies difficult in vivo. Therefore, the need exists for in vitro models of gallbladder epithelium. We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice. Cells were grown in a monolayer on porous inserts over a feeder layer of fibroblasts. These nontransformed cells can be successively passaged and maintain a well-differentiated epithelial cell phenotype as shown by morphologic criteria, characterized by polarized columnar epithelial cells with prominent microvilli and intercellular junctions. Organotypic cultures showed columnar cells simulating in vivo morphology. This culture system should be valuable in delineating cellular processes relating to CFTR in gallbladder epithelium.     

ALGAL‐RELATED TASTES AND ODORS IN PHOENIX WATER SUPPLY: PRELIMINARY REPORT

Frequent episodes of algal‐related tastes and odors (T & O) in drinking waters in metropolitan Phoenix, Arizona prompted initiation of a three‐year project in July 1999 to investigate the occurrence of T & O metabolites and to develop a comprehensive management strategy to reduce the problems in drinking water supplies in arid environments. Two metabolites, 2‐methylisoborneol (MIB) and geosmin, have been identified as compounds responsible for the earthy‐musty tastes and odors in water supplies. Both were detected in the water supply system, including source rivers, reservoirs, canal delivery system and water treatment plants. Higher concentrations of MIB and geosmin occurred in distribution canals than in the upstream reservoirs indicating that significant production of the T & O compounds occurs within the canal system. A baseline‐monitoring program has been established for the complex water supply system, with special emphasis on the canal system. Efforts are underway to investigate possible correlations between physical/chemical parameters, algal composition and biomass, with the occurrence of MIB and geosmin. In addition, several physical and chemical treatments are planned for the canal system to reduce algal growth and related MIB and geosmin concentrations.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

MtDNA diversity of the critically endangered Mekong giant catfish (Pangasianodon gigas Chevey, 1913) and closely related species: implications for conservation

Catfishes of the family Pangasiidae are an important group that contributes significantly to the fisheries of the Mekong River basin. In recent times the populations of several catfish species have declined, thought to be due to overfishing and habitat changes brought about by anthropogenic influences. The Mekong giant catfish Pangasianodon gigas Chevey, 1913 is listed as Critically Endangered on the IUCN Red List. In the present study, we assessed the level of genetic diversity of nine catfish species using sequences of the large subunit of mitochondrial DNA (16S rRNA). Approximately 570 base pairs (bp) were sequenced from 672 individuals of nine species. In all species studied, haplotype diversity and nucleotide diversity ranged from 0.118±0.101 to 0.667±0.141 and from 0.0002±0.0003 to 0.0016±0.0013, respectively. Four haplotypes were detected among 16 samples from natural populations of the critically endangered Mekong giant catfish. The results, in spite of the limited sample size for some species investigated, indicated that the level of genetic variation observed in wild populations of the Mekong giant catfish (haplotype diversity=0.350±0.148, nucleotide diversity=0.0009±0.0008) is commensurate with that of some other related species. This finding indicates that (1) wild populations of the Mekong giant catfish might be more robust than currently thought or (2) present wild populations of this species carry a genetic signature of the historically larger population(s). Findings from this study also have important implications for conservation of the Mekong giant catfish, especially in designing and implementing artificial breeding programme for restocking purposes.     

Peptides as tools and drugs for immunotherapies.

Peptides are essential tools for discovery and pre-clinical and pharmaceutical development of viral and cancer vaccines ('active immunotherapies') as well as for therapeutic antibodies ('passive immunotherapies'). They help to trigger and analyze immune responses at a molecular level (B-cell, T-helper and CTL epitopes). They contribute largely to the design of new vaccine candidates and to the generation of monoclonal antibodies. They are also valuable analytical reference compounds for the structural characterisation by liquid chromatography and mass spectrometry of recombinant proteins used as biopharmaceuticals. As for other therapeutic applications, formulation, solubilisation, batch consistency and stability, issues have to be addressed to allow the pre-clinical and clinical development of this class of compounds as immunotherapeutic drugs. In the present review, three case studies dealing with (i) the design and the characterisation of Respiratory Syntycial Virus subunit vaccines, (ii) peptide-based melanoma vaccines, and (iii) therapeutic monoclonal antibodies, all investigated in clinical trials, are reported and discussed.     

Purification and characterization of a dextranase from Sporothrix schenckii

A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 °C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the K _m for soluble dextran was 0.067 ± 0.003% (w/v). Salts of Hg²⁺, (UO₂)²⁺, Pb²⁺, Cu²⁺, and Zn²⁺ inhibited by affecting both V _max and K _m. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 °C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 °C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 °C. All of these isolates grew on glucose at 35 °C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature. Received: 29 December 1997 / Accepted: 4 March 1998