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Objective. Chronic stress (CS)-induced abnormal metabolism and other subsequent aspects of abnormality are threatening human health. Little is known regarding whether and how protein post-translational-modifications (PTMs) correlate with abnormal metabolism under CS. The aim of this study was to address this issue and also identify novel key protein PTM. Methods. First, we screened which pan-PTM had significant change between control and CS female mice and whether clinical CS females had similar pan-PTM change. Second, we performed quantitative PTM-omics and metabolomics to verify the correlation between abnormal protein PTMs and atypical metabolism. Third, we performed quantitative phospho-omics to identify the key PTM-regulating enzyme and investigate the interaction between PTM protein and PTM-regulating enzyme. Fourth, we attempted to rectify the abnormal metabolism by correcting the activity of the PTM-regulating enzyme. Finally, we examined whether the selected key protein was also correlated with stress scores and atypical metabolism in clinical women. Results. We initially found that multiple tissues of CS female mice have downregulated pan-crotonylation, and verified that the plasma of clinical CS females also had downregulated pan-crotonylation. Then we determined that ATP5O-K51 crotonylation decreased the most and also caused gross ATP5O decrement, whereas the plasma of CS mice had downregulated phospholipids. Next, downregulating ATP5O crotonylation partially recapitulated the downregulated phospholipid metabolism in CS mice. Next, we verified that HDAC2-S424 phosphorylation determined its decrotonylation activity on ATP5O-K51. Furthermore, correcting HDAC2 hyper-phosphorylation recovered the gross ATP5O level and partially rescued the downregulated phospholipid metabolism in CS mice. Finally, the ATP5O level was also significantly lower and correlated with high stress scores and downregulated phospholipid metabolism in clinical female plasma. Conclusion. This study discovered a novel PTM mechanism involving two distinct types of PTM in CS and provided a novel reference for the clinical precautions and treatments of CS., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Liang-Jian Chen et al.) more...

Summary Tobacco smoke exposure has obvious and complex effects on the immune system of the human upper respiratory tract, including pro-inflammatory and anti-immune effects. Exposure to tobacco smoke is closely related to the occurrence and development of allergic rhinitis, the common rhinitis and sinusitis. The innate immune system is influenced by tobacco smoking through its effects on the respiratory mucosa and its adjuncts, natural killer cells, dendritic cells, neutrophils and innate immune receptors. Cigarette smoke can also affect the humoral immunity and cellular immunity, altering the acquired immune condition of the upper respiratory tract. Tobacco smoke exposure promotes the occurrence and development of the upper respiratory tract infectious diseases and allergic diseases by changing the composition of microflora in the upper respiratory tract., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.) more...

Kainic acid (KA)-induced neuronal death is linked to mitochondrial dysfunction and ER stress. Melatonin is known to protect hippocampal neurons from KA-induced apoptosis, but the exact mechanisms underlying melatonin protective effects against neuronal mitochondria disorder and ER stress remain uncertain. In this study, we investigated the sheltering roles of melatonin during KA-induced apoptosis by focusing on mitochondrial dysfunction and ER stress mediated signal pathways. KA causes mitochondrial dynamic disorder and dysfunction through calpain activation, leading to neuronal apoptosis. Ca 2+ chelator BAPTA-AM and calpain inhibitor calpeptin can significantly restore mitochondrial morphology and function. ER stress can also be induced by KA treatment. ER stress inhibitor 4-phenylbutyric acid (PBA) attenuates ER stress-mediated apoptosis and mitochondrial disorder. It is worth noting that calpain activation was also inhibited under PBA administration. Thus, we concluded that melatonin effectively inhibits KA-induced calpain upregulation/activation and mitochondrial deterioration by alleviating Ca 2+ overload and ER stress. more...

Background & Objective: Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1)is a kind of transmembrane glycoprotein,which is induced by epidermal growth factor (EGF),and develops inhibitory negative feedback by specific binding with epidermal growth factor receptor (EGFR). This research was to explore the molecular mechanism of LRIG1 inhibiting EGFR signal pathway., Methods: Plasmid p3XFLAG-CMV9-LRIG1 was transfected into neuroglioma cell line H4. Changes of LRIG1 and EGFR expression at mRNA and protein levels were measured by RT-PCR and Western blot, respectively. Changes of cell proliferation and apoptosis were analyzed by MTT and flow cytometry methods., Results: LRIG1 mRNA level in p3XFLAG-CMV9-LRIG1 transfected H4 cells (1.997+/-0.114) was significantly higher than that in control H4 cells (0.500+/-0.031),and p3XFLAG-CMV9 transfected group (0.357+/-0.035) (P< 0.001). LRIG1 protein level in p3XFLAG-CMV9-LRIG1 transfected H4 cells (1.790+/-0.119) was significantly higher than that in control H4 cells (0.717+/-0.038, P< 0.001), and p3XFLAG-CMV9 transfected H4 cells (0.930+/-0.076, P=0.001). EGFR mRNA level in p3XFLAG-CMV9-LRIG1 transfected H4 cells (0.463+/-0.033) was significantly lower than that in control H4 cells (1.157+/-0.067, P< 0.001), and p3XFLAG-CMV9 transfected H4 cells (0.933+/-0.058, P=0.002). EGFR protein level in p3XFLAG-CMV9-LRIG1 transfected H4 cells (0.703+/-0.067) was significantly lower than that in control H4 cells (1.280+/-0.078, P=0.003),and p3XFLAG-CMV9 transfected H4 cells (1.163+/-0.068,P=0.009). Apoptosis rate in LRIG1-transfected H4 cells (18.89%)was lower than that in control H4 cells (3.11%), and vector-transfected H4 cells (5.42%, P< 0.001)., Conclusion: LRIG1 participates in construction of negative feedback loop of EGFR, which may inhibits growth of glioma cells. more...

Previous studies from our laboratory revealed a novel protein of 38.5 kD on the surface of malignant cell lines of hematopoetic origin that exhibit susceptibility to naive natural killer (NK) cell-mediated lysis. In contrast, p38.5 was not detected on the surface of NK cell-resistant carcinoma cell lines or normal cells. We now report that this protein is differentially expressed, intracellularly, in malignant cell lines of both hematopoetic and epithelial origin compared with nonmalignant cells. To characterize p38.5 further, we used a previously developed antipeptide antibody (anti-11-mer) to probe cDNA expression libraries and subsequently performed 5' extension by rapid amplification of cDNA ends (RACE). Sequence analyses of these cDNA clones reveal open reading frames (ORFs) that include the previously identified 11-mer peptide from purified, native p38.5 and that have identical sequences to a gene of unknown function on chromosome 19. Nucleotide sequence data obtained from these cDNA clones, as well as analysis of the genomic sequence, permitted design of primers for reverse transcriptase-polymerase chain reaction (RT-PCR) that resulted in a cDNA clone encoding an ORF of 361 amino acids; the clone was identical to a sequence encoded by an unpublished mRNA in GenBank. Anti-p38.5 antibody against the 11-mer peptide encoded in exon 5 and against a 25-mer peptide encoded in exon 1 both reacted with the same protein in immunoprecipitation studies, providing further evidence of identity. RT-PCR and Northern blot analyses both demonstrated p38.5 gene transcripts in normal cells, nonmalignant cell lines and malignant cell lines of epithelial as well as hematopoietic origin. Semiquantitative studies revealed a greater level of p38.5 gene transcription in malignant cell lines compared with nonmalignant cells. Immunoblot analyses of protein expression confirmed and extended the latter studies by revealing substantially greater levels of the 38.5 kD protein in whole cell extracts of malignant cell lines compared with nonmalignant cells. Quantitative differences in detection of the 38.5 kD protein and mRNA in NK susceptible- hematopoietic malignancies compared with NK resistant-carcinomas were not observed. These experiments suggest that the p38.5 gene (Haymaker) is widely expressed in human cells of different tissue origins but that elevated expression is associated with the malignant phenotype., (Copyright 2001 Wiley-Liss, Inc.) more...

A receptor-ligand interaction exclusive to natural killer (NK) cell-mediated recognition and triggering of tumor cell destruction has not yet been identified. In contrast, molecules that are involved in cellular adhesion and regulation of NK cytolysis have been well studied. In this report, a novel tumor surface protein is identified that exhibits characteristics of a recognition structure for naive NK cells. A tagged ligand-cell adsorption technique revealed a 38.5-kD plasma membrane protein (p38.5) from a prototypical NK-susceptible cell line (K562) that preferentially bound to NK cells (CD3(-)CD5(-)CD16(+)) relative to T lymphocytes (CD3(+)CD5(+) CD16(-)). The molecule was purified to apparent homogeneity for further characterization. An amino acid sequence of an 11-mer internal peptide of p38.5 did not exhibit homology to known proteins. Affinity-purified antibody generated against this peptide (anti-p38.5) reacted with a single protein of 38.5 kD on Western blots of whole cell extracts of K562. Flow cytometry and immunoprecipitation studies of surface-labeled tumor cells demonstrated expression of p38.5 on NK-susceptible tumor cell lines (K562, MOLT-4, Jurkat), whereas p38.5 was not detected on NK-resistant tumor cell lines (A549, Raji, MDA-MB-231). Significantly, p38.5 loss variants derived from wild-type Jurkat and Molt-4 cell lines exhibited decreased susceptibility to NK cell-mediated lysis demonstrating a strong association between cell surface expression of p38.5 and cytotoxicity. Purified p38.5 retained preferential binding to NK cells and inhibited NK activity in a dose-dependent manner, thereby providing direct evidence of a role in the lytic process. Binding studies identified a 70-kD membrane protein from NK cells as a possible receptor for the p38.5 tumor ligand. Consistent with cellular adsorption studies, the 70-kD, p38.5 binding protein was not detected on T lymphocytes. Based on studies demonstrating selective binding of p38.5 to NK cells, lack of expression on NK-resistant tumor cell lines and ability of the purified molecule to block cytolysis, we conclude that p38.5 may serve as a recognition/triggering ligand for naive human NK cells. more...

Rhodamine 123 (R123) is a mitochondria-specific prototype anticancer agent because its target is the energy-producing mechanism of the cell. The goal of this study was to investigate the relationship between intracellular R123 accumulation and cytotoxicity in a R123-sensitive cell line (RS) and a R123-resistant subline (RR) that we developed. Cytotoxicity after exposure to R123 (0-60 micrograms/ml) was assessed using the clonogenic assay. Intracellular R123 was extracted with acid-alcohol and measured by fluorimetry. The rate of R123 accumulation over 1 hr was significantly higher (P less than 0.0001) for RS cells (4.65 +/- 0.39 micrograms/min/10(6) cells) than for RR cells (1.29 +/- 0.24 micrograms/min/10(6) cells). R123 accumulation in RS cells was strongly correlated (r = 0.80; P less than 0.0001) with cytotoxicity. Treatment of RR cells with verapamil (100 microM) reversed R123 resistance. The resulting dose-survival curve was identical to the dose-response curve of RS cells treated with R123 alone. Cellular content of R123 in RR cells treated with verapamil increased to a level similar to that of RS cells and correlated with cytotoxicity. These data suggest that cytotoxicity of R123 in B16 cells results from increased cellular accumulation of R123. more...