Inhibition of Endoplasmic Reticulum Stress-induced Apoptosis by Silkworm Storage Protein 1

The endoplasmic reticulum (ER) plays essential roles indispensable for cellular activity and survival, including functions such as protein synthesis, secretory and membrane protein folding, and Ca²⁺ release in cells. The ER is sensitive to stresses that can lead to the aggregation and accumulation of misfolded proteins, which eventually triggers cellular dysfunction; severe or prolonged ER stress eventually induces apoptosis. ER stress-induced apoptosis causes several devastating diseases such as atherosclerosis, neurodegenerative diseases, and diabetes. In addition, the production of biopharmaceuticals such as monoclonal antibodies requires the maintenance of normal ER functions to achieve and maintain the production of high-quality products in good quantities. Therefore, it is necessary to develop methods to efficiently relieve ER stress and protect cells from ER stress-induced apoptosis. The silkworm storage protein 1 (SP1) has anti-apoptotic activities that inhibit the intrinsic mitochondrial apoptotic pathway. However, the role of SP1 in controlling ER stress and ER stress-induced apoptosis has not been investigated. In this paper, we demonstrate that SP1 can inhibit apoptosis induced by a well-known ER stress inducer, thapsigargin, by alleviating the decrease in cell viability and mitochondrial membrane potential. Interestingly, SP1 significantly blocked increases in CHOP and GRP78 expression as well as ER Ca2+ leakage into the cytosol following ER stress induction. This indicates that SP1 protects cells from ER stressinduced apoptosis by functioning as an upstream inhibitor of apoptosis. Therefore, studying SP1 function can offer new insights into protecting cells against ER stress-induced apoptosis for future applications in the biopharmaceutical and medicine industries.     

Stable Formamidinium‐Based Perovskite Solar Cells via In Situ Grain Encapsulation

Formamidinium (FA)‐based lead iodide perovskites have emerged as the most promising light‐absorber materials in the prevailing perovskite solar cells (PSCs). However, they suffer from the phase‐instability issue in the ambient atmosphere, which is holding back the realization of the full potential of FA‐based PSCs in the context of high efficiency and stability. Herein, the tetraethylorthosilicate hydrolysis process is integrated with the solution crystallization of FA‐based perovskites, forming a new film structure with individual perovskite grains encapsulated by amorphous silica layers that are in situ formed at the nanoscale. The silica not only protects perovskite grains from the degradation but also enhances the charge‐carrier dynamics of perovskite films. The underlying mechanism is discussed using a joint experiment‐theory approach. Through this in situ grain encapsulation method, PSCs show an efficiency close to 20% with an impressive 97% retention after 1000‐h storage under ambient conditions.     

The ATPase Pathway That Drives the Kinesin-14 Kar3Vik1 Powerstroke

Kar3, a Saccharomyces cerevisiae microtubule minus-end-directed kinesin-14, dimerizes with either Vik1 or Cik1. The C-terminal globular domain of Vik1 exhibits the structure of a kinesin motor domain and binds microtubules independently of Kar3 but lacks a nucleotide binding site. The only known function of Kar3Vik1 is to cross-link parallel microtubules at the spindle poles during mitosis. In contrast, Kar3Cik1 depolymerizes microtubules during mating but cross-links antiparallel microtubules in the spindle overlap zone during mitosis. A recent study showed that Kar3Vik1 binds across adjacent microtubule protofilaments and uses a minus-end-directed powerstroke to drive ATP-dependent motility. The presteady-state experiments presented here extend this study and establish an ATPase model for the powerstroke mechanism. The results incorporated into the model indicate that Kar3Vik1 collides with the microtubule at 2.4 μm⁻¹ s⁻¹ through Vik1, promoting microtubule binding by Kar3 followed by ADP release at 14 s⁻¹. The tight binding of Kar3 to the microtubule destabilizes the Vik1 interaction with the microtubule, positioning Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is associated with rotation of the coiled-coil stalk, and the postpowerstroke ATP hydrolysis at 26 s⁻¹ is independent of Vik1, providing further evidence that Vik1 rotates with the coiled coil during the powerstroke. Detachment of Kar3Vik1 from the microtubule at 6 s⁻¹ completes the cycle and allows the motor to return to its initial conformation. The results also reveal key differences in the ATPase cycles of Kar3Vik1 and Kar3Cik1, supporting the fact that these two motors have distinctive biological functions.     

Genotype patterns and characteristics of PRNP in the Korean population

Creutzfeldt-Jakob disease (CJD), included in the human transmissible spongiform encephalopathies (TSE), is widely known to be caused by an abnormal accumulation of misfolding prion protein in the brain. Human prion protein gene (PRNP) is mapped in chromosome 20p13 and many single nucleotide polymorphisms (SNPs) in PRNP have been discovered. However, the functionality of SNPs in PRNP is yet unclear, though several SNPs have been known as important mutation related with susceptibility human prion diseases. Our aim is to identify specific genotype patterns and characteristics in the PRNP genomic region and to understand susceptibility among Korean discriminated prion disease patients, suspected CJD patients and the KARE data group. Here, we have researched genotypes and SNPs allele frequencies in PRNP in discriminated prion disease patients group (n = 22), suspected prion diseases patients group (n = 163) and the Korea Association REsource (KARE) data group (n = 296) in Korea. The sequencing regions were promoter region, exon1 and exon2 with their junction parts among 481 samples. A total of 25 SNPs were shown in this study. Nucleotide frequencies of all SNPs are exceedingly tended to bias toward dominant homozygote types except in rs2756271. Genotype frequencies at codon 129 and 219 coding region were similar with previous studies in Korea and Japan. Pathogenic mutations such as 102P/L, 200E/K and 203V/I were observed in discriminated CJD patients group, and 180V/I and 232M/R were shown in suspected prion disease patients group and the KARE data group. A total of 10 SNPs were newly identified, six in the promoter region, one in exon 2 and three in the 3′ UTR. The strong and unique linkage disequilibrium (D' = 0.94, r² = 0.89) was observed between rs57633656 and rs1800014 which is located in codon 219 coding region. We expect that these data can be provided to determine specific susceptibility and a protective factor of prion diseases not only in Koreans but also in East Asians.     

Cdk5与大鼠脑缺血再灌注损伤时细胞凋亡的关系

目的利用大鼠可逆性大脑中动脉栓塞模型（MCAO）,研究周期素依赖性蛋白激酶5（cyclin dependent kinase5,Cdk5）对视网膜母细胞瘤蛋白（Rb）的磷酸化作用与细胞凋亡的关系。方法线栓法制作大鼠MCAO模型,随机分为再灌注0、3、6、9、24h组。通过免疫组化染色观察缺血侧大脑Cdk5和磷酸化Rb的数量和变化,TUNEL标记法检测凋亡神经元数量和变化,Western blot检测不同再灌注时间Cdk5蛋白水平的表达。结果缺血侧cdk5随再灌注时间增加而增加,24h达到高峰;再灌注6h出现磷酸化的视网膜母细胞瘤蛋白（pRb）,并随再灌注时间增加而增加,24h达到高峰。缺血侧凋亡阳性细胞变化趋势与Cdk5基本一致。缺血侧Cdk5蛋白的表达随再灌注时间增加而增加,缺血对侧无明显变化。结论大鼠局限性脑缺血再灌注损伤可以诱导Cdk5数量和激酶活性增加,通过底物磷酸化作用引起神经细胞凋亡。     

Curcumin ameliorates TNF-α-induced ICAM-1 expression and subsequent THP-1 adhesiveness via the induction of heme oxygenase-1 in the HaCaT cells

Adhesion molecules such as ICAM-1 are important in the infiltration of leukocytes into the site of inflammation. In this study, we investigated the inhibitory effects of curcumin on ICAM-1 expression and monocyte adhesiveness as well as its underlying action mechanism in the TNF-α-stimulated keratinocytes. Curcumin induced expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. In addition, curcumin induced Nrf2 activation in dose- and time-dependent manners in the HaCaT cells. Curcumin suppressed TNF-α- induced ICAM-1 expression and subsequent monocyte adhesion, which were reversed by the addition of tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, or HO-1 knockdown using siRNA. Furthermore, Nrf2 knockdown using siRNA reversed the inhibitory effect of curcumin on the TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that curcumin may exert its anti-inflammatory activity by suppressing the TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes. [BMB Reports 2013;46(8): 410-415]     

Viral metagenomics reveals diverse viruses in the fecal samples of children with diarrhea

Diarrhea is the third leading cause of death in developing countries in children under the age of five. About half a million children die of diarrhea every year, most of which in developing countries. Viruses are the main pathogen of diarrhea. In China, the fecal virome of children with diarrhea has been rarely studied. Using an unbiased viral metagenomics approach, we analyzed the fecal virome in children with diarrhea. Many DNA or RNA viruses associated with diarrhea identified in those fecal samples were mainly from six families of Adenoviridae, Astroviridae, Caliciviridae, Parvoviridae, Picornaviridae, and Reoviridae. Among them, the family of Caliciviridae accounts for the largest proportion of 78.42%, following with Adenoviridae (8.94%) and Picornaviridae (8.36%). In addition to those diarrhea-related viruses that have already been confirmed to cause human diarrhea, the viruses not associated with diarrhea were also identified including anellovirus and picobirnavirus. This study increased our understanding of diarrheic children fecal virome and provided valuable information for the prevention and treatment of viral diarrhea in this area.     

Fine mapping of the circling (cir) gene on the distal portion of mouse chromosome 9

Circling mice manifest profound deafness, head-tossing, and bi-directional circling behavior, which they inherit in autosomal recessive manner. Histologic examination of the inner ear reveals abnormalities of the region around the organ of Corti, spiral ganglion neurons, and outer hair cells. A genetic linkage map was constructed for an intraspecific backcross between cir and C57BL/6J mice. The cir gene was mapped to a region between D9Mit116/D9Mit15 and D9Mit38 on mouse chromosome (Chr) 9. Estimated distances between cir and D9Mit116, and between cir and D9Mit38 were 0.70 +/- 0.40 and 0.23 +/- 0.23 cM, respectively. Order of the markers was defined as follows: centromere - D9Mit182 - D9Mit51/D9Mit79/D9Mit310 - D9Mit212/D184 - D9Mit116/D9Mit15 - cir - D9Mit38 - D9Mit20 - D9Mit243 - D9Mit16 - D9Mit55/D9Mit125 - D9Mit281. On the basis of genetic mapping, we constructed a yeast artificial chromosome (YAC) contig across the cir region. The cir gene is located between the lactotransferrin (ltf) and microtubule-associated protein (map4) genes. The distal portion of mouse Chr 9 encompassing the cir region is homologous with human chromosome 3p21, which contains the Deafness, form B: Autosomal Recessive Deafness (DFNB6) locus. Therefore, the circling mouse is a potential animal model for DFNB6 deafness in humans.     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.     

Association between polymorphisms of matrix metalloproteinase 11 (MMP-11) and Kawasaki disease in the Korean population

AimsThis study was conducted to investigate the associations between single nucleotide polymorphisms (SNPs) of matrix metalloproteinases (MMPs) and Kawasaki disease (KD) in the Korean population.Main methodsA total 0f 101 KD patients and 306 healthy controls were examined. MMP7 (rs10502001, G/A, Arg77His), MMP11 (rs738792, T/C, Ala38Val), MMP12 (rs652438, A/G, Ile357Val) and MMP26 (rs2499953, A/G, Lys43Glu) genes were genotyped from the genomic DNA using direct sequencing. The results were then analyzed using logistic regression models, adjusting for gender as covariates.Key findingsThe four SNPs were in Hardy–Weinberg equilibrium. Only the MMP11 polymorphism (rs738792) was associated with KD. The SNP (rs738792) showed a statistically significant association with KD in the codominant (OR = 1.61, 95% CI = 1.11–2.34, P = 0.011) and dominant (OR = 1.92, 95% CI = 1.21–3.06, P = 0.006) models. However, there was no association between polymorphisms of other MMP genes and KD.SignificanceOverall, the results of this study indicate that MMP11 polymorphism may be associated with KD in the Korean population.