Pigment Cell & Melanoma Research
·
2025
·Vol. 38
·期 5
·e70048
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IF 2.6
Yingzi Zhang,Teng Liu,Qingsong Yang,Qiaorong Huang,Jiayi Ai,Yefeng Yuan,Wei Li,Aihua Wei
The diagnostic approaches for Hermansky-Pudlak Syndrome (HPS) include genetic sequencing, immunoblotting, electron microscopy (EM), and flow cytometry with mepacrine staining. However, these methods are often impractical for routine clinical use due to high cost, technical complexity, and limited availability. In this study, we evaluated dense granules (DGs) function in HPS mouse models using flow cytometry with mepacrine and FluoZin-3 staining. We then developed a standardized, practical flow cytometry-based protocol and validated it in patients with HPS and oculocutaneous albinism (OCA), which were confirmed by whole-mount EM. In HPS mouse models (BLOC-1, BLOC-2, BLOC-3, and AP-3 deficient mutants), mepacrine uptake was consistently reduced. FluoZin-3 fluorescence showed subtype-specific zinc dysregulation, with elevated levels in BLOC-1, BLOC-2, and AP-3 mutants but decreased levels in the BLOC-3 mutant. In contrast, the OCA-6 mouse mutant showed no significant changes in either mepacrine or FluoZin-3 uptake. Similar patterns were observed in HPS and non-syndromic OCA patients. Our findings indicate that the protocol can enable the precise diagnosis and preliminary subtype classification of HPS, while also facilitating differential diagnosis between HPS and OCA. This method offers a rapid, clinically accessible alternative to conventional diagnostic techniques and may also be applicable to other storage pool disorders with DG defects.
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产品关联
DOI
PubMed
高影响因子
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
·
2015
·Vol. 56
·期 13
·8166-8178
·
IF 3.404
Guoxu Xu,Daohuan Kang,Chaoyang Zhang,Hui Lou,Chen Sun,Qian Yang,Lixia Lu,Guo-Tong Xu,Jingfa Zhang,Fang Wang
Purpose:Zinc transporter 8 (ZnT8) was downregulated in hypoxic retina, which could be rescued by hypoxia-inducible factor-1α (HIF-1α) inhibition. Erythropoietin (EPO) protects retinal cells in diabetic rats through inhibiting HIF-1α as one of its mechanisms. We hence tried to explore the effect of EPO in regulating ZnT8 and protecting retinal cells in diabetic rats and possible mechanisms.Methods:Diabetes was induced in Sprague-Dawley rats. Intravitreal injection of EPO was performed 1 month after diabetes onset. The CoCl2-treated rat Müller cell line (rMC-1) was cotreated with EPO, soluble EPO receptor (sEPOR), digoxin, or U0126. Cell viability, cell death, and intracellular zinc level were examined. The expression of ZnT8, HIF-1α, AKT, and ERK was studied.Results:In diabetic rat retinas, EPO significantly decreased HIF-1α expression and increased ZnT8 expression. In CoCl2-treated rMC-1 cells, EPO increased cell viability and decreased intracellular zinc. Erythropoietin or digoxin could activate ERK pathway, downregulate HIF-1α, and upregulate ZnT8. The effect of EPO was abolished by sEPOR and U0126. Transient knockdown of ZnT8 increased intracellular zinc level, but not to a degree that would decrease cell viability or cause cell death.Conclusions:In diabetic retinas, EPO maintains zinc homeostasis through activating the ERK pathway and downregulating HIF-1α, and thus upregulating ZnT8 expression. This work proposed a possible new protective mechanism for EPO in, and indicated a potential target for, the treatment of diabetic retinopathy.
研究领域:
眼科学
·
内分泌学
·
细胞生物学
·
分子生物学
·
糖尿病研究
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DOI
PubMed
高影响因子
PLoS One
·
2015
·Vol. 10
·期 7
·e0133451
·
IF 3.234
Wen-jie Zhao,Qun Song,Zi-jin Zhang,Li Mao,Wei-juan Zheng,Xin Hu,Hong-zhen Lian
Zinc, an essential trace element, is involved in many important physiological processes. Cell responses to zinc stress show time-dependent effects besides concentration-dependence and tissue-specificity. Herein, we investigated the time-dependent differential expression of the proteome in A549 cells after administered with ZnSO 4 for both 9 and 24 h using 2DE. 123 differentially expressed protein spots were detected, most of which were up-regulated by Zn 2+ treatment. Interestingly, 49 proteins exhibited significant differential expression repeatedly during these two treatment periods, and moreover showed a conserved change with different ratios and four time-dependent expression patterns. Pattern 1 (up-regulated with rapid initial induction and subsequent repression) and pattern 4 (down-regulated with steady repression) were the predominant expression patterns. The abundances of the proteins in patterns 1 and 4 after 24 h of zinc treatment are always lower than that after 9 h, indicating that exogenous zinc reduced the expression of proteins in cells after 24 h or longer. Importantly, these findings could also reflect the central challenge in detecting zinc homeostasis proteins by 2DE or other high throughput analytical methods resulting from slight variation in protein expression after certain durations of exogenous zinc treatment and/or low inherent protein content in cells. These time-dependent proteome expression patterns were further validated by measuring dynamic changes in protein content in cells and in expression of two proteins using the Bradford method and western blotting, respectively. The time-dependent changes in total zinc and free Zn 2+ ion contents in cells were measured using ICP-MS and confocal microscopy, respectively. The kinetic process of zinc homeostasis regulated by muffling was further revealed. In addition, we identified 50 differentially expressed proteins which are predominantly involved in metabolic process, cellular process or developmental process, and function as binding, catalytic activity or structural molecule activity. This study further elucidates our understanding of dynamic nature of the cellular response to zinc stress and the mechanism of zinc homeostasis.
研究领域:
细胞生物学
·
蛋白质组学
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分子生物学
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毒理学
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PubMed
高影响因子
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