Yoo Hun Noh

Activation of PERK Signaling Attenuates Aβ-Mediated ER Stress

Alzheimers disease (AD) is characterized by the deposition of aggregated beta-amyloid (Aβ), which triggers a cellular stress response called the unfolded protein response (UPR). The UPR signaling pathway is a cellular defense system for dealing with the accumulation of misfolded proteins but switches to apoptosis when endoplasmic reticulum (ER) stress is prolonged. ER stress is involved in neurodegenerative diseases including AD, but the molecular mechanisms of ER stress-mediated Aβ neurotoxicity still remain unknown. Here, we show that treatment of Aβ triggers the UPR in the SK-N-SH human neuroblastoma cells. Aβ mediated UPR pathway accompanies the activation of protective pathways such as Grp78/Bip and PERK-eIF2α pathway, as well as the apoptotic pathways of the UPR such as CHOP and caspase-4. Knockdown of PERK enhances Aβ neurotoxicity through reducing the activation of eIF2α and Grp8/Bip in neurons. Salubrinal, an activator of the eIF2α pathway, significantly increased the Grp78/Bip ER chaperone resulted in attenuating caspase-4 dependent apoptosis in Aβ treated neurons. These results indicate that PERK-eIF2α pathway is a potential target for therapeutic applications in neurodegenerative diseases including AD.

Kynurenic acid attenuates MPP(+)-induced dopaminergic neuronal cell death via a Bax-mediated mitochondrial pathway.

Kynurenic acid (KYNA), a tryptophan metabolite in the kynurenine pathway, is protective against various insults. However, the molecular mechanism of this protective effect has not been identified. In this study, we examined the protective effects of KYNA against 1-methyl-4-phenylpyridinium (MPP(+)), the best-characterized toxin inducing pathological changes resembling Parkinsons disease (PD), using SH-SY5Y and SK-N-SH human neuroblastoma cells. Pre-treatment of KYNA attenuated MPP(+)-induced neuronal cell death in SH-SY5Y and SK-N-SH cells. MPP(+)-induced cell death was preceded by increases in Bax expression and mitochondrial dysfunction, such as collapse of mitochondrial membrane potential (DeltaPsi(m)), release of cytochrome c from mitochondria into the cytoplasm, and increases in caspase-9/-3 activities. KYNA effectively inhibited all of these mitochondrial apoptotic processes. Our results indicate that KYNA plays a protective role by down-regulating Bax expression and maintaining mitochondrial function in MPP(+)-induced neuronal cell death, and suggest that KYNA may have therapeutic potential in PD.

Vitamin D Status and Vitamin D Receptor Gene Polymorphisms Are Associated with Pelvic Floor Disorders in Women

Objectives To investigate if vitamin D receptor (VDR) gene polymorphisms and circulating vitamin D levels are associated with pelvic floor disorders (PFDs). Methods In this case-control study, 25-hydroxy-vitamin D (25[OH]D) serum levels were analyzed in 47 females with PFDs and 87 healthy females (controls), respectively. The VDR gene polymorphisms were determined by using polymerase chain reaction and performing digestions with 4 restriction enzymes i.e., ApaI, TaqI, FokI, and BsmI. Vitamin D levels of patients were divided into <20 ng/mL, 20 to 30 ng/mL, and ≥30 ng/mL categories. Results Our correlative analysis of VDR polymorphisms as a function of the presence of PFD showed that ApaI and BsmI polymorphisms were significantly associated with PFD in vitamin-D-deficiency and insufficiency groups (P < 0.05). Mean vitamin D levels did not differ between the PFD case (13.01 ± 0.84 ng/mL) and control (15.11 ± 1.04 ng/mL) groups (P > 0.05). However, there was a significant difference in the distribution of vitamin D levels between study group and controls using Pearsons χ2 test (<20 ng/mL, 20–30 ng/mL, and >30 ng/mL: 87.2%, 12.8%, and 0% in the study group and 75.9%, 16.1%, and 8.0% in controls, respectively, P < 0.05). Taken together, our observations suggest that vitamin D levels could be associated with PFDs and that 2 polymorphisms (i.e., ApaI and BsmI) in the VDR gene may contribute to an increased prevalence of PFDs in women with insufficient levels of vitamin D. Conclusions Examining vitamin D levels and performing a VDR genotype analysis may be helpful for assessing PFD risk.

Baculovirus titration method based on MOI values for optimizing recombinant protein expression of the anti-cancer vaccine candidate GA733-Fc using Sf9 insect cells

The baculovirus expression system has been considered as a highly efficient tool for the production of recombinant biopharmaceutical proteins. The recombinant antigenic glycoprotein GA733 is a cell surface protein that is strongly expressed in human colorectal cancer. Efficient virus titration should be established to achieve optimal multiplicity of infection (MOI) conditions, which are in turn essential for strong expression of the recombinant GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) in the baculovirus‐insect system. In the present study, the Sf9 cell line was transfected with plasmid DNA containing the GA733‐Fc expression cassette under the control of the baculovirus polyhedron promoter. MOI values (0.05, 0.1, 0.5, 1, and 3) were calculated based on both microscope observations and results of titration assay and then used to determine the optimum recombinant expression and harvested sample [cell culture media (CM) or cell lysate (CL)]. The pFastBac dual vector carrying the GA733‐Fc gene was constructed to express GA733‐Fc and used to generate recombinant baculoviruses. Western blotting results showed that recombinant protein expression was dependent on the MOI. In addition, CM and CL showed significant differences in protein synthesis and protein secretion capacities. Our findings suggested that our proposed titration method can be used for reliable calculation of MOI values, which significantly influence recombinant GA733‐Fc protein expression in the baculovirus‐insect cell system.