Neural tissue is arisen from presumptive ectoderm via inhibition of bone morphogenetic protein (BMP) signaling during Xenopus early development. Previous studies demonstrate that ectopic expression of dominant negative BMP4 receptor (DNBR) produces neural tissue in animal cap explants (AC) and also increases the expression level of various genes involved in neurogenesis. To investigate detail mechanism of neurogenesis in transcriptional level, we analyzed RNAs increased by DNBR using total RNA sequencing analysis and identified several candidate genes. Among them, xCITED2 (Xenopus CBP/p300-interacting transcription activator) was induced 4.6 fold by DNBR and preferentially expressed in neural tissues at tadpole stage. Ectopic expression of xCITED2 induced anterior neural genes without mesoderm induction and reduced BMP downstream genes, an eye specific marker and posterior neural marker. Taken together, these results suggest that xCITED2 may have a role in the differentiation of anterior neural tissue during Xenopus early development.
Animals ; Bone Morphogenetic Proteins ; Ectoderm ; Embryonic Structures ; Eye ; Larva ; Mesoderm ; Neurogenesis ; RNA ; Sequence Analysis, RNA ; Xenopus

Phagocytosis of serum- and IgG-opsonized zymosan (SOZ and IOZ, respectively) particles into J774A.1 macrophages induced apoptosis of the cells, accompanied by the expression of p21(WAF1), one of cyclin-dependent protein kinase (CDK) inhibitors. Furthermore, phagocytosis of SOZ and IOZ particles into macophages induced superoxide formation. Tat-superoxide dismutase (SOD), which is readily transduced into the cells using Tat-domain, protected the cells from the apoptosis induced by phagocytosis of SOZ and IOZ particles. lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) also caused the apoptosis of the cells. However, Tat-SOD could not protect the cells from LPS/IFN-gamma induced apoptosis, suggesting that apoptosis mechanisms involved are different from each other. In the present study, we determined the amounts of nitric oxide (NO) produced by SOZ, IOZ, and LPS/IFN-gamma, and found that SOZ and IOZ did not induce the generation of NO in macrophages, whereas LPS/ IFN-gamma did. The apoptosis due to phagocytosis was accompanied with the release of cytochrome c from mitochondrial membrane to cytosolic fraction. Furthermore, SOZ and IOZ induced the cleavage of procasapase-3 (35 kDa) to give rise to an active caspase-3 (20 kDa), which was blocked by Tat- SOD but not by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of NO. On the other hand, LPS/IFN-gamma caused the activation of procaspase-3, which was blocked by PTIO but not by Tat-SOD. Taken together, phagocytosis of SOZ and IOZ particles induced apoptosis through superoxide but not NO in macrophages, accompanied with the release of cytochrome c and the activation of caspase-3.
Apoptosis/*immunology ; Caspases/metabolism ; Cell Line ; Cyclins/biosynthesis ; Cytochromes c/metabolism ; Immunoglobulin G/*immunology ; Interferon Type II/pharmacology ; Lipopolysaccharides/pharmacology ; Macrophages/*immunology/metabolism ; Nitric Oxide/*metabolism ; Opsonins/immunology ; Phagocytosis/*physiology ; Superoxide Dismutase/metabolism ; Superoxides/*metabolism ; Zymosan

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.

The inducible 70 kDa heat shock proteins (Hsp70) in mice are encoded by two almost identical genes, hsp70.1 and hsp70.3. Studies have found that only hsp70.1 is induced by hypertonic stress while both hsp70.1 and hsp70.3 genes are expressed in response to heat shock stress. It is unclear if the human counterparts, hsp70-2 and hsp70-1, are differentially regulated by heat shock and osmotic stress. This study found that only hsp70-2 was induced by hypertonic stress in human embryonic kidney epithelial cells and fibroblasts, while heat shock stress induced both hsp70-1 and hsp70-2. The human hsp70-2 promoter region contains three TonE (tonicity-responsive enhancer) sites, which were reported to play an important role in the response to hypertonicity. When the reporter plasmids containing different parts of the 5' flanking region of hsp70-2 were transfected into human embryonic kidney epithelial cells or fibroblasts, one TonE site at -135 was found to play a key role in the response to hypertonicity. The inactivation of the TonE site using site-directed mutagenesis led to the complete loss of induction by hypertonicity, which demonstrates the essential role of the TonE site. This suggests that the TonE site and the TonEBP (TonE binding protein) are the major regulators for the cellular response against high osmolarity in human kidney tissue.
Transcription, Genetic/drug effects/genetics ; Transcription Factors/genetics/*physiology ; Saline Solution, Hypertonic/*pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Protein Binding ; Promoter Regions (Genetics)/genetics ; Point Mutation ; Mutagenesis, Site-Directed ; Humans ; HSP70 Heat-Shock Proteins/*genetics/metabolism ; Gene Expression Regulation/*drug effects ; DNA-Binding Proteins/genetics/metabolism ; Cell Line ; Binding Sites/genetics ; Base Sequence ; 5' Flanking Region/genetics

The epidermis is a physiological barrier to protect organisms against environment. During the aging process, skin tissues undergo various changes including morphological and functional changes. The transcriptional regulation of genes is part of cellular reaction of aging process. In order to examine the changes of gene expression during the aging process, we used the primary cell culture system of human keratinocytes. Since UV radiation is the most important environmental skin aggressor, causing skin cancer and other problems including premature skin aging, we examined the changes of gene expression in human keratinocytes after UV irradiation using oligonucleotide microarray containing over 10,000 genes. We also compared the gene expression patterns of the senescent and UV treated cells. Expression of the variety of genes related to transcription factors, cell cycle regulation, immune response was altered in human keratinocytes. Some of down-regulated genes are represented in both senescent and UV treated cells. The results may provide a new view of gene expression following UVB exposure and aging process in human keratinocytes.
Aging* ; Cell Cycle ; Epidermis ; Gene Expression* ; Humans* ; Keratinocytes* ; Oligonucleotide Array Sequence Analysis ; Primary Cell Culture ; Skin ; Skin Aging ; Skin Neoplasms ; Transcription Factors

The heterodimeric c-Jun/c-Fos, an activator protein-1 (AP-1) has been implicated in mesoderm induction (Dong et al., 1996; Kim et al., 1998) whereas the homodimer of c-Jun was reported to be involved in neural inhibition during the early development of Xenopus embryos. During the early vertebrate development AP-1 involvement in the neural induction is still not clearly understood. We report here that AP-1 has a role in Zic3 expression, a critical proneural gene and a primary regulator of neural and neural crest development (Nakata et al., 1997; Nakata et al., 1998). AP-1 was able to induce the Zic3 gene in a dose dependent manner but other homo- or hetero-dimeric proteins, such as c-Jun/c-Jun, JunD/FosB or JunD/Fra-1 were not. The inhibition of AP-1 activity using morpholino antisenses of c-jun mRNAs blocked the Zic3 expression induced by activin. In addition, co-injection of c-jun mRNA rescued the down-regulated Zic3 expression. The promoter region of isolated Zic3 genomic DNA was found to possess several consensus-binding site of AP-1. Thus, in the functional assays, AP-1 could increase promoter activity of Zic3 gene. These findings suggest that proneural gene, Zic3 may be regulated by heterodimeric AP-1(c-Jun/c-Fos) and it may have a role in activin signaling for the regulation of neural specific gene, Zic3.
Activins/pharmacology/physiology ; Animals ; Base Sequence ; Binding Sites/genetics ; Consensus Sequence/genetics ; Dimerization ; Embryo, Nonmammalian/metabolism ; *Gene Expression Regulation, Developmental ; Homeodomain Proteins/*genetics ; Molecular Sequence Data ; Promoter Regions (Genetics)/genetics ; Proto-Oncogene Proteins c-fos/genetics/physiology ; Proto-Oncogene Proteins c-jun/genetics/physiology ; RNA, Antisense/genetics ; Research Support, Non-U.S. Gov't ; Transcription Factor AP-1/genetics/*physiology ; Transcription Factors/*genetics ; *Transcription, Genetic ; Up-Regulation ; Xenopus Proteins/*genetics ; Xenopus laevis/*embryology/*genetics

Background/Aims: The utility of serum pepsinogen (sPG) I and the sPGI/II ratio as biomarkers for screening individuals with gastric cancer (GC) has not been established in Korea. The aim of this study was to define the role of sPG, especially sPGII, in GC screening. Methods: This study enrolled 2,940 subjects, including patients with GC (n=1,124) or gastric dysplasia (n=353) and controls (n=1,463). Tests to determine sPG levels and Helicobacter pylori (HP) infection status were performed. Area under the curve and receiver operating characteristic curve were calculated to identify the optimal cutoff values for sPG. The usefulness of sPG levels for the detection of GC and gastric dysplasia was validated by multivariate logistic regression. Results: The sPGI/II ratio was associated with the risk of gastric dysplasia and advanced-stage intestinal-type GC (IGC). In contrast, sPGII was associated with the risk of early-stage diffuse-type GC (DGC). Significantly higher risk was indicated by an sPGI/II ratio <3 for gastric dysplasia and advanced-stage IGC and by sPGII levels ≥20 µg/L for early-stage DGC. Positive HP status showed a stronger association with DGC than with IGC. When sPGII level and HP status were combined, the prevalence of DGC was higher in the ≥20 µg/L sPGII and HP-positive group. Age younger than 40 years was strongly related to early-stage DGC, especially in females (odds ratio, 21.00; p=0.006). Conclusions sPGII ≥20 ng/mL and positive HP status suggest a risk of early-stage DGC, particularly in young adult females in South Korea.

BACKGROUND: Intra-class correlation coefficients (ICCs) provide a statistical means of testing the reliability. However, their interpretation is not well documented in the orthopedic field. The purpose of this study was to investigate the use of ICCs in the orthopedic literature and to demonstrate pitfalls regarding their use. METHODS: First, orthopedic articles that used ICCs were retrieved from the Pubmed database, and journal demography, ICC models and concurrent statistics used were evaluated. Second, reliability test was performed on three common physical examinations in cerebral palsy, namely, the Thomas test, the Staheli test, and popliteal angle measurement. Thirty patients were assessed by three orthopedic surgeons to explore the statistical methods testing reliability. Third, the factors affecting the ICC values were examined by simulating the data sets based on the physical examination data where the ranges, slopes, and interobserver variability were modified. RESULTS: Of the 92 orthopedic articles identified, 58 articles (63%) did not clarify the ICC model used, and only 5 articles (5%) described all models, types, and measures. In reliability testing, although the popliteal angle showed a larger mean absolute difference than the Thomas test and the Staheli test, the ICC of popliteal angle was higher, which was believed to be contrary to the context of measurement. In addition, the ICC values were affected by the model, type, and measures used. In simulated data sets, the ICC showed higher values when the range of data sets were larger, the slopes of the data sets were parallel, and the interobserver variability was smaller. CONCLUSIONS: Care should be taken when interpreting the absolute ICC values, i.e., a higher ICC does not necessarily mean less variability because the ICC values can also be affected by various factors. The authors recommend that researchers clarify ICC models used and ICC values are interpreted in the context of measurement.
Adolescent ; Biomedical Research/*methods/*standards ; Cerebral Palsy ; Child ; Child, Preschool ; Computer Simulation ; Databases, Factual ; Female ; Humans ; Male ; Models, Theoretical ; Orthopedics/*methods/*standards ; Physical Examination ; Range of Motion, Articular ; Reproducibility of Results ; Research Design ; Statistics as Topic ; Young Adult

Purpose: Delayed images may not be acquired due to severe pain, drowsiness, or worsening vital signs while waiting after blood pool imaging in three-phase bone scintigraphy. If the hyperemia in the blood pool image contains information from which increased uptake on the delayed images can be inferred, the generative adversarial network (GAN) can generate the increased uptake from the hyperemia. We attempted to apply pix2pix, a type of conditional GAN, to transform hyperemia into increased bone uptake. Methods: We enrolled 1464 patients who underwent three-phase bone scintigraphy for inflammatory arthritis, osteomyelitis, complex regional pain syndrome (CRPS), cellulitis, and recent bone injury. Blood pool images were acquired 10 min after intravenous injection of Tc-99 m hydroxymethylene diphosphonate, and delayed bone images were obtained after 3 h.The model was based on the open-source code of the pix2pix model with perceptual loss. Increased uptake in the delayed images generated by the model was evaluated using lesion-based analysis by a nuclear radiologist in areas consistent with hyperemia in the blood pool images. Results: The model showed sensitivities of 77.8% and 87.5% for inflammatory arthritis and CRPS, respectively. In osteomyelitis and cellulitis, their sensitivities of about 44% were observed. However, in cases of recent bone injury, the sensitivity was only 6.3% in areas consistent with focal hyperemia. Conclusion The model based on pix2pix generated increased uptake in delayed images matching the hyperemia in the blood pool image in inflammatory arthritis and CRPS.

Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.
Animals ; Butyrates ; Cyclic N-Oxides ; Embryonic Structures ; Hand ; Imidazoles ; Neurites ; Neurons ; Nitric Oxide ; Nitric Oxide Synthase ; PC12 Cells ; Rats ; RNA, Messenger