Autophagy and its implication in Chinese hamster ovary cell culture

Chinese hamster ovary (CHO) cells, that are widely used for production of therapeutic proteins, are subjected to apoptosis and autophagy under the stresses induced by conditions such as nutrient deprivation, hyperosmolality and addition of sodium butyrate. To achieve a cost-effective level of production, it is important to extend the culture longevity. Until now, there have been numerous studies in which apoptosis of recombinant CHO (rCHO) cells was inhibited, resulting in enhanced production of therapeutic proteins. Recently, autophagy in rCHO cells has drawn attention because it can be genetically and chemically controlled to increase cell survival and productivity. Autophagy is a global catabolic process which involves multiple pathways and genes that regulate the lysosomal degradation of intracellular components. A simultaneous targeting of anti-apoptosis and pro-autophagy could lead to more efficient protection of cells from stressful culture conditions. In this regard, it is worthwhile to have a detailed understanding of the autophagic pathway, in order to select appropriate genes and chemical targets to manage autophagy in rCHO cells, and thus to enhance the production of therapeutic proteins.     

Expression of a lipase on the cell-surface of Escherichia coli using the OmpW anchoring motif and its application to enantioselective reactions

Microbial-surface display is the expression of proteins or peptides on the surface of cells by fusing an appropriate protein as an anchoring motif. Here, the outer membrane protein W (OmpW) was selected as a fusion partner for functional expression of Pseudomonas fluorescence SIK W1 lipase (TliA) on the cell-surface of Escherichia coli. Localization of the truncated OmpW-TliA fusion protein on the cell-surface was confirmed by immunoblotting and functional assay of lipase activity. Enantioselective hydrolysis of rac-phenylethyl butanoate by the displayed lipase resulted in optically active (R)-phenyl ethanol with 96 % enantiomeric excess and 44 % of conversion in 5 days. Thus, a small outer membrane protein OmpW, is a useful anchoring motif for displaying an active enzyme of ~50 kDa on the cell-surface and the surface-displayed lipase can be employed as an enantioselective biocatalyst in organic synthesis.     

Rhamnetin and Cirsiliol Induce Radiosensitization and Inhibition of Epithelial-Mesenchymal Transition (EMT) by miR-34a-mediated Suppression of Notch-1 Expression in Non-small Cell Lung Cancer Cell Lines

Radioresistance is a major cause of decreasing the efficiency of radiotherapy for non-small cell lung cancer (NSCLC). To understand the radioresistance mechanisms in NSCLC, we focused on the radiation-induced Notch-1 signaling pathway involved in critical cell fate decisions by modulating cell proliferation. In this study, we investigated the use of Notch-1-regulating flavonoid compounds as novel therapeutic drugs to regulate radiosensitivity in NSCLC cells, NCI-H1299 and NCI-H460, with different levels of radioresistance. Rhamnetin and cirsiliol were selected as candidate Notch-1-regulating radiosensitizers based on the results of assay screening for activity and pharmacological properties. Treatment with rhamnetin or cirsiliol reduced the proliferation of NSCLC cells through the suppression of radiation-induced Notch-1 expression. Indeed, rhamnetin and cirsiliol increased the expression of tumor-suppressive microRNA, miR-34a, in a p53-dependent manner, leading to inhibition of Notch-1 expression. Consequently, reduced Notch-1 expression promoted apoptosis through significant down-regulation of the nuclear factor-κB pathway, resulting in a radiosensitizing effect on NSCLC cells. Irradiation-induced epithelial-mesenchymal transition was also notably attenuated in the presence of rhamnetin and cirsiliol. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing and epithelial-mesenchymal transition inhibition effects of rhamnetin and cirsiliol we observed in vitro. In these mice, tumor volume was significantly reduced by combinational treatment with irradiation and rhamnetin or cirsiliol compared with irradiation alone. Taken together, our findings provided evidence that rhamnetin and cirsiliol can act as promising radiosensitizers that enhance the radiotherapeutic efficacy by inhibiting radiation-induced Notch-1 signaling associated with radioresistance possibly via miR-34a-mediated pathways.     

The Sulfonylurea Receptor 1 (Sur1)-Transient Receptor Potential Melastatin 4 (Trpm4) Channel

The sulfonylurea receptor 1 (Sur1)-NC_Ca-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC_Ca-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K_ATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.     

Characterization of Fibrinogen Binding by Glycoproteins Srr1 and Srr2 of Streptococcus agalactiae

The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of K_D = 2.1 × 10⁻⁵ and 3.7 × 10⁻⁶ m, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6–8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a “dock, lock, and latch” mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.     

Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.     

Dongia rigui sp. nov., isolated from freshwater of a large wetland in Korea

A motile, curved to twisted rod-shaped aerobic bacterium, designated strain 04SU4-P^T, was isolated from freshwater collected from the Woopo wetland (Republic of Korea). Cells were observed to be Gram-stain negative, catalase negative and oxidase positive. The major fatty acids (>10 % of the total) were identified as C_19:0 ω8c cyclo (24.6 %), C_16:0 (24.3 %) and C_18:1 ω7c (13.1 %). The DNA G+C content was determined to be 71.5 mol%. The major polar lipids were identified as phosphatidylglycerol, phosphatidylethanolamine, one unknown phospholipid and one unknown aminolipid. The major ubiquinone was determined to be Q-10. A phylogenetic tree based on 16S rRNA gene sequences showed that strain 04SU4-P^T forms an evolutionary lineage within the genus Dongia and its nearest neighbour is Dongia mobilis LM22^T (98.0 % sequence similarity). Genomic DNA–DNA hybridization of stain 04SU4-P^T with D. mobilis LM22^T showed relatedness of only 34.2 %. The phenotypic characteristics indicate the strain 04SU4-P^T can be distinguished from the sole member of the genus Dongia. On the basis of the data presented in this study, strain 04SU4-P^T represents a novel species, for which the name Dongia rigui is proposed. The type strain is 04SU4-P^T (KCTC 23341^T = JCM 17521^T).     

N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.     

Therapeutic and pharmacokinetic characterizations of an anti-amyloidogenic bis-styrylbenzene derivative for Alzheimer’s disease treatment

Alzheimer’s disease drug discovery regarding exploration into the molecules and processes has focused on the intrinsic causes of the brain disorder correlated with the accumulation of amyloid-β. An anti-amyloidogenic bis-styrylbenzene derivative, KMS80013, showed excellent oral bioavailability (F = 46.2%), facilitated brain penetration (26%, iv) in mouse and target specific in vivo efficacy in acute AD mouse model attenuating the cognitive deficiency in Y-maze test. Acute toxicity (LD₅₀ >2000 mg/kg) and hERG channel inhibition (14% at 10 μM) results indicated safety of KMS80013.     

Nematicidal activity of Lysobacter capsici YS1215 and the role of gelatinolytic proteins against root-knot nematodes

Lysobacter capsici YS1215 is a soil-borne strain that could inhibit the growth of phytopathogenic fungi, including Phytophthora capsici, Rhizoctonia solani and Fusarium oxysporum, as well as root-knot nematodes. The effect of different concentrations of bacterial culture filtrate (BCF) of L. capsici YS1215 on the mortality of second-stage juveniles (J2) of Meloidogyne incognita was studied using 24-well plates. The J2 mortality increased with increasing concentrations of BCF. YS1215 also produces gelatinases in the culture filtrate. To study its role in nematicidal activities, the partial purification and the characterisation of gelatinolytic proteins were done from the culture medium of the YS1215. The partially purified proteins showed three clear bands with molecular weights estimated using zymography to be 255.7, 232.1 and 146.4 kDa. The optimal pH and temperature for the proteins were 8.0 and 40°C, respectively. The activity of the proteins was inhibited by ethylenediaminetetraacetic acid, FeCl₃ and 1,10-phenanthroline, whereas it was activated by MnCl₂. The proteins may belong to the group of metalloproteases. Moreover, the proteins could hydrolyse skimmed milk, collagen, gelatin and bovine serum albumin (BSA) as substrates, but not casein. The proteins could induce 75% J2 mortality in five days and degrade the J2 bodies. The present study demonstrates the role of the gelatinolytic proteins in the nematicidal potential of L. capsici YS1215.