Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential protein-protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.     

Salivary PYY: a putative bypass to satiety

Peptide YY(3-36) is a satiation hormone released postprandially into the bloodstream from L-endocrine cells in the gut epithelia. In the current report, we demonstrate PYY(3-36) is also present in murine as well as in human saliva. In mice, salivary PYY(3-36) derives from plasma and is also synthesized in the taste cells in taste buds of the tongue. Moreover, the cognate receptor Y2R is abundantly expressed in the basal layer of the progenitor cells of the tongue epithelia and von Ebner's gland. The acute augmentation of salivary PYY(3-36) induced stronger satiation as demonstrated in feeding behavioral studies. The effect is mediated through the activation of the specific Y2 receptor expressed in the lingual epithelial cells. In a long-term study involving diet-induced obese (DIO) mice, a sustained increase in PYY(3-36) was achieved using viral vector-mediated gene delivery targeting salivary glands. The chronic increase in salivary PYY(3-36) resulted in a significant long-term reduction in food intake (FI) and body weight (BW). Thus this study provides evidence for new functions of the previously characterized gut peptide PYY(3-36) suggesting a potential simple and efficient alternative therapeutic approach for the treatment of obesity.     

Diversity of the abundant pKLC102/PAGI-2 family of genomic islands in Pseudomonas aeruginosa

The known genomic islands of Pseudomonas aeruginosa clone C strains are integrated into tRNA(Lys) (pKLC102) or tRNA(Gly) (PAGI-2 and PAGI-3) genes and differ from their core genomes by distinctive tetranucleotide usage patterns. pKLC102 and the related island PAPI-1 from P. aeruginosa PA14 were spontaneously mobilized from their host chromosomes at frequencies of 10% and 0.3%, making pKLC102 the most mobile genomic island known with a copy number of 30 episomal circular pKLC102 molecules per cell. The incidence of islands of the pKLC102/PAGI-2 type was investigated in 71 unrelated P. aeruginosa strains from diverse habitats and geographic origins. pKLC102- and PAGI-2-like islands were identified in 50 and 31 strains, respectively, and 15 and 10 subtypes were differentiated by hybridization on pKLC102 and PAGI-2 macroarrays. The diversity of PAGI-2-type islands was mainly caused by one large block of strain-specific genes, whereas the diversity of pKLC102-type islands was primarily generated by subtype-specific combination of gene cassettes. Chromosomal loss of PAGI-2 could be documented in sequential P. aeruginosa isolates from individuals with cystic fibrosis. PAGI-2 was present in most tested Cupriavidus metallidurans and Cupriavidus campinensis isolates from polluted environments, demonstrating the spread of PAGI-2 across habitats and species barriers. The pKLC102/PAGI-2 family is prevalent in numerous beta- and gammaproteobacteria and is characterized by high asymmetry of the cDNA strands. This evolutionarily ancient family of genomic islands retained its oligonucleotide signature during horizontal spread within and among taxa.     

Alpha-oxo semicarbazone peptide or oligodeoxynucleotide microarrays

We describe in this paper the preparation and characterization of semicarbazide glass slides and their use for the fabrication of microarrays using site-specific alpha-oxo semicarbazone ligation. The functional density and homogeneity of the semicarbazide glass slides were optimized by analyzing the reactivity of the layer toward a synthetic glyoxylyl fluorescent probe. Oligonucleotide microarrays were prepared by site-specific immobilization of glyoxylyl oligodeoxynucleotides. The slides were directly used in the hybridization assays using fluorescence detection and displayed a significant gain in sensibility as compared to the aldehyde glass slide/amino oligodeoxynucleotide chemistry. Semicarbazide slides were also used for the immobilization of a biotinylated peptide alpha-oxo aldehyde. The peptide microarrays allowed model interaction studies with streptavidin or an anti-biotin antibody.     

Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II

Pokeweed antiviral protein II (PAPII), a 30 kDa protein isolated from leaves of Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. The protein sequence of PAPII shows only 41% identity to PAP and PAP-S, two other antiviral proteins isolated from pokeweed. We isolated a cDNA corresponding to PAPII and introduced it into tobacco plants. PAPII expressed in transgenic tobacco was correctly processed to the mature form as in pokeweed and accumulated to at least 10-fold higher levels than wild-type PAP. We had previously observed a significant decrease in transformation frequency with PAP and recovered only two transgenic lines expressing 1–2 ng per mg protein. In contrast, eight different transgenic lines expressing up to 250 ng/mg PAPII were recovered, indicating that PAPII is less toxic than PAP. Two symptomless transgenic lines expressing PAPII were resistant to tobacco mosaic virus, potato virus X and the fungal pathogen Rhizoctonia solani. The level of viral and fungal resistance observed correlated well with the amount of PAPII protein accumulated. Pathogenesis-related protein PR1 was constitutively expressed in transgenic lines expressing PAPII. Although PR1 was constitutively expressed, no increase in salicylic acid levels was detected, indicating that PAPII may elicit a salicylic acid-independent signal transduction pathway.     

Overexpression of superoxide dismutase or glutathione peroxidase protects against the paraquat + maneb-induced Parkinson disease phenotype

Oxidative stress has been implicated in the pathogenesis of Parkinson disease based on its role in the cascade of biochemical changes that lead to dopaminergic neuronal death. This study analyzed the role of oxidative stress as a mechanism of the dopaminergic neurotoxicity produced by the combined paraquat and maneb model of the Parkinson disease phenotype. Transgenic mice overexpressing either Cu,Zn superoxide dismutase or intracellular glutathione peroxidase and non-transgenic mice were exposed to saline, paraquat, or the combination of paraquat + maneb twice a week for 9 weeks. Non-transgenic mice chronically exposed to paraquat + maneb exhibited significant reductions in locomotor activity, levels of striatal dopamine and metabolites, and dopaminergic neurons in the substantia nigra pars compacta. In contrast, no corresponding effects were observed in either Cu,Zn superoxide dismutase or glutathione peroxidase transgenic mice. Similarly, the increase in levels of lipid hydroperoxides in the midbrain and striatum of paraquat + maneb-treated non-transgenic mice was not detected in either Cu,Zn superoxide dismutase or glutathione peroxidase transgenic mice. To begin to determine critical pathways of paraquat + maneb neurotoxicity, the functions of cell death-inducing and protective mechanisms were analyzed. Even a single injection of paraquat + maneb in the non-transgenic treated group modulated several key pro- and anti-apoptotic proteins, including Bax, Bad, Bcl-xL, and upstream stress-induced cascade. Collectively, these findings support the assertion that protective mechanisms against paraquat + maneb-induced neurodegeneration could involve modulation of the level of reactive oxygen species and alterations of the functions of specific signaling cascades.     

Non-monotonic Response to Monotonic Stimulus: Regulation of Glyoxylate Shunt Gene-Expression Dynamics in Mycobacterium tuberculosis

Understanding how dynamical responses of biological networks are constrained by underlying network topology is one of the fundamental goals of systems biology. Here we employ monotone systems theory to formulate a theorem stating necessary conditions for non-monotonic time-response of a biochemical network to a monotonic stimulus. We apply this theorem to analyze the non-monotonic dynamics of the σ^B-regulated glyoxylate shunt gene expression in Mycobacterium tuberculosis cells exposed to hypoxia. We first demonstrate that the known network structure is inconsistent with observed dynamics. To resolve this inconsistency we employ the formulated theorem, modeling simulations and optimization along with follow-up dynamic experimental measurements. We show a requirement for post-translational modulation of σ^B activity in order to reconcile the network dynamics with its topology. The results of this analysis make testable experimental predictions and demonstrate wider applicability of the developed methodology to a wide class of biological systems.