Nramp1 and Other Transporters Involved in Metal Withholding during Infection

During the course of infection, many natural defenses are set up along the boundaries of the host-pathogen interface. Key among these is the host response to withhold metals to restrict the growth of invading microbes. This simple act of nutritional warfare, starving the invader of an essential element, is an effective means of limiting infection. The physiology of metal withholding is often referred to as “nutritional immunity,” and the mechanisms of metal transport that contribute to this host response are the focus of this review.     

Targeting of the mouse Slc39a2 (Zip2) gene reveals highly cell-specific patterns of expression, and unique functions in zinc, iron, and calcium homeostasis

Fourteen members of the Slc39a superfamily of metal ion uptake transporters have been identified in mice and humans, but the physiological functions of most remain obscure. Herein, we created mice with Zip2 (Slc39a2) genes in which the open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP), to study temporal and spatial patterns of Zip2 gene expression and examine the physiological roles of this transporter. Expression of this gene was remarkably cell-type specific and developmentally regulated in pericentral hepatocytes, developing keratinocytes, and a subset of immature dendritic cells in the immune system. In addition, the Zip2 gene was transiently expressed in giant trophoblast cells in the placenta. Although the Zip2 gene was not essential under conditions of normal dietary zinc, it played an important role in adapting to dietary zinc deficiency during pregnancy, and in the homeostasis of iron in the liver as well as iron and calcium in developing embryos. These studies suggest that active expression of the Zip2 gene in these few specific cell types, aforementioned, plays a particularly important role during zinc deficiency. These studies further reveal novel interactions between zinc transporter function and the homeostasis of other essential metals.     

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

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Zinc nutritional status influences ZnT1 and ZIP4 gene expression in children with a high risk of zinc deficiency

IntroductionSubclinical deficiency of zinc is associated with impairment of immune system function, growth, and cognitive development in children. Although plasma zinc is the best available biomarker of the risk of zinc deficiency in populations, its sensitivity for early detection of deficiency is limited. Therefore, we aimed to investigate zinc deficiency among preschool children and its relationship with whole blood gene expression of zinc transporters ZIP4 and ZnT1.Material and methodsThis cross-sectional study included 139 children aged 32–76 months enrolled in philanthropic day-care centers. We performed an anthropometric evaluation, weighed food record and dietary record for dietary assessment, blood sample collection for zinc, and whole blood gene expression analyses of ZnT1 (SLC30A1) and ZIP4 (SLC39A4).ResultsZinc deficiency was observed in 26.6 % of the children despite adequate zinc intake and a phytate:zinc molar ratio < 18. Usual zinc intake did not affect whole blood gene expression of zinc transporters, but zinc status influenced ZnT1 and ZIP4 whole blood mRNA. Children with zinc deficiency exhibited 37.1 % higher ZnT1 expression and 45.3 % lower ZIP4 expression than children with adequate zinc (p < 0.05).ConclusionChildren with plasma zinc deficiency exhibited higher expression of ZnT1 and lower expression of ZIP4 in whole blood mRNA, reinforcing the existence of strong regulation of mineral homeostasis according to the nutritional status, indicating that this analysis may be useful in the evaluation of dietary interventions.     

Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.     

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.     

The relationship between beta cell activation and SLC30A8/ZnT8 levels of the endocrine pancreas and maternal zinc deficiency in rats

ObjectivesZinc, which is found in high concentrations in the β-cells of the pancreas, is also a critical component for the endocrine functions of the pancreas. SLC30A8/ZnT8 is the carrier protein responsible for the transport of zinc from the cytoplasm to the insulin granules. The aim of this study was to investigate how dietary zinc status affects pancreatic beta cell activation and ZnT8 levels in infant male rats born to zinc-deficient mothers.MethodsThe study was performed on male pups born to mothers fed a zinc-deficient diet. A total of 40 male rats were divided into 4 equal groups. Group 1: In addition to maternal zinc deficiency, this group was fed a zinc-deficient diet. Group 2: In addition to maternal zinc deficiency, this group was fed a standard diet. Group 3: In addition to maternal zinc deficiency, this group was fed a standard diet and received additional zinc supplementation. Group 4: Control group. Pancreas ZnT8 levels were determined by ELISA method and insulin-positive cell ratios in β-cells by immunohistochemistry.ResultsThe highest pancreatic ZnT8 levels and anti-insulin positive cell ratios in the current study were obtained in Group 3 and Group 4. In our study, the lowest pancreatic ZnT8 levels were obtained in Group 1 and Group 2, and the lowest pancreatic anti-insulin positive cell ratios were obtained in Group 1.ConclusionThe results of the present study; in rats fed a zinc-deficient diet after maternal zinc deficiency has been established shows that ZnT8 levels and anti-insulin positive cell ratios in pancreatic tissue, which is significantly suppressed, reach control values with intraperitoneal zinc supplementation.     

The zebrafish Znt1asa17 mutant reveals roles of zinc transporter-1a in embryonic development

BackgroundZinc is one of the vital micronutrients required through various developmental stages in animals. Zinc transporter-1 (ZnT1; Slc30a1) is essential in vertebrates for nutritional zinc uptake and cellular zinc extrusion. Knockout of ZnT1 is lethal in vertebrates and there are therefore few functional studies of this protein in vivo.MethodsIn the present study we characterised the embryonic development in a zebrafish Znt1a mutant (Znt1a^sa17) which is lacking the last 40 amino acids of Znt1a as generated by TILLING. In parallel experiments, we compared the development of a zebrafish embryo Znt1a morphant (Znt1a^MO) which was generated by knockdown of Znt1a using morpholino-modified oligonucliotides.ResultsThe homozygous Znt1a^sa17 embryo is viable, but displays a subtle phenotype informing on the biological roles of Znt1a. The Znt1a^sa17 fish have delayed development, including attenuated epiboly. They further show a decrease in phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2), retarded yolk resorption, and impaired clearance of free Zn²⁺ from the vitelline fluid and its storage in hatching gland cells. All these aberrations are milder versions of those observed upon knockdown of Znt1a by morpholinos. Interestingly, the phenotype could be rescued by addition of the cell-permeable zinc chelator, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) to the incubation medium and was aggravated by addition of zinc(II). Thus, the Znt1a^sa17 mutant has a reduced ability to handle zinc and can be characterised as a hypomorph.ConclusionThis study is the first to show that the last 40 amino acids of Znt1a are of importance for its role in zinc homeostasis and ability to activate the MAPK/ERK pathway contrary to what was previously thought.     

An overview of a wide range of functions of ZnT and Zip zinc transporters in the secretory pathway

Zinc plays essential roles in the early secretory pathway for a number of secretory, membrane-bound, and endosome/lysosome-resident enzymes. It enables the enzymes to fold properly and become functional, by binding as a structural or catalytic component. Moreover, zinc secreted from the secretory vesicles/granules into the extracellular space has a pivotal role as a signaling molecule for various physiological functions. Zinc transporters of the Slc30a/ZnT and Slc39a/Zip families play crucial roles in these functions, mediating zinc influx to and efflux from the lumen of the secretory pathway, constitutively or in a cell-specific manner. This paper reviews current knowledge of the ways these two zinc transporters perform these tasks by manipulating zinc homeostasis in the secretory pathway. Recent questions concerning zinc released into the cytoplasm from the secretory pathway, which then functions as an intracellular signaling molecule, are also briefly reviewed, emphasizing zinc transporter functions.     

人α降钙素基因相关肽基因在酵母细胞中的分泌表达与产物的分离纯化

化学合成的人α降钙素基因相关肽(CCRP)基因用PCR法改造后使其能正确融合在酵母分泌型表达载体pVT102U/α中的α交配因子前导肽序列之后,然后进行克隆并转化酵母宿主菌S－78进行表达．培养物的上清用酶标(ELlSA)鉴定为阳性,而对照S－78、pVT102u／α为阴性,表达量用ELISA定量大于2mg／L。表达产物经阳离子交按层析(CM—Sphadex C₂₅)和HPLC纯化得到了HPLC纯产品。纯化后的CGRP能引起小鼠血压的降低,说明表达的目的蛋白既有CGRP的免疫结合活性,又有CGRP的生理活性。测定其N－端10个氨基酸序列,证明人工合成的CGRP基因在酵母细胞中已正确表达。     

Distribution of Secretory Pathway Ca 2+ ATPase (SPCA1) in Neuronal and Glial Cell Cultures

1. Secretory pathway Ca²⁺ ATPase type 1 (SPCA1) is a newly recognized Ca²⁺/Mn²⁺-transporting pump localized in membranes of the Golgi apparatus.2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues.3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains.4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells.5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically.6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, α-tubulin or MBP.7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells.8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.     

Wnt5a及其信号通路与血管新生的研究进展

Wnt5a是Wnt蛋白家族中的成员之一,在细胞成熟、胚胎发育等过程中发挥着重要作用。研究表明Wnt5a的表达调控及其信号通路与血管新生密切相关,并且在血管新生性相关疾病中发挥了重要作用。本文从Wnt5a与其相关信号转导通路对血管新生的影响以及分子机制等方面进行阐述和展望,旨在为以Wnt5a为靶点进行血管新生性疾病的防治提供理论依据。     

Association of the solute carrier family 11 member 1 gene polymorphisms with susceptibility to leprosy in a Brazilian sample

Natural resistance-associated macrophage protein 1/solute carrier family 11 member 1gene (Nramp1/Slc11a1) is a gene that controls the susceptibility ofinbred mice to intracellular pathogens. Polymorphisms in the humanSlc11a1/Nramp1 gene have been associated with host susceptibilityto leprosy. This study has evaluated nine polymorphisms of theSlc11a1/Nramp1 gene [(GT)n, 274C/T, 469+14G/C, 577-18G/A, 823C/T,1029 C/T, 1465-85G/A, 1703G/A, and 1729+55del4] in 86 leprosy patients (67 and 19patients had the multibacillary and the paucibacillary clinical forms of the disease,respectively), and 239 healthy controls matched by age, gender, and ethnicity. Thefrequency of allele 2 of the (GT)n polymorphism was higher in leprosy patients [p =0.04, odds ratio (OR) = 1.49], whereas the frequency of allele 3 was higher in thecontrol group (p = 0.03; OR = 0.66). Patients carrying the 274T allele (p= 0.04; OR = 1.49) and TT homozygosis (p = 0.02; OR = 2.46), suchas the 469+14C allele (p = 0.03; OR = 1.53) of the 274C/T and 469+14G/Cpolymorphisms, respectively, were more frequent in the leprosy group. The leprosy andcontrol groups had similar frequency of the 577-18G/A, 823C/T, 1029C/T, 1465-85G/A,1703G/A, and 1729+55del4 polymorphisms. The 274C/T polymorphism in exon 3 and the469+14G/C polymorphism in intron 4 were associated with susceptibility to leprosy,while the allele 2 and 3 of the (GT)n polymorphism in the promoter region wereassociated with susceptibility and protection to leprosy, respectively.     

Insight into the structures of the second and fifth transmembrane domains of Slc11a1 in membrane mimics

Slc11a1 is an integral membrane protein with 12 putative transmembrane domains and functions as a pH‐coupled divalent metal cation transporter. In the present study, the structures of the peptides corresponding to the second and fifth transmembrane domains of Slc11a1 (from 88 to 109 for TMD2 and from 190 to 215 for TMD5) were determined in membrane‐mimic environments by CD and NMR techniques. It was demonstrated that TMD2 and TMD5 form an α‐helical structure in 30% 2,2,2‐trifluoroethanol (TFE) and 40% hexafluoro‐2‐propanol (HFIP) aqueous solution, respectively. The α‐helix of TMD5 displays a less space‐occupied face consisting of the residues Ala194, Gly197, Thr201, Ala204 and Gly208. The α‐helix is partially unfolded in the N‐terminal region when Gly197 is substituted by Val. The unfolding of the helix in the N‐terminal part and/or increase in volume at the less space‐occupied face of the helix may exert an effect on the arrangement of TMD5 in membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

血清miR-26a、miR-130a与老年急性脑梗死患者PI3K/Akt信号通路和预后的关系研究

摘要 目的：研究血清miR-26a、miR-130a与老年急性脑梗死（ACI）患者磷酸酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）信号通路和预后的关系。方法：选取2020年3月-2022年10月我院收治的98例老年ACI患者作为研究组,同期选取60例于本院体检健康的老年人作为对照组。采用实时荧光定量聚合酶链式反应（qRT-PCR）检测血清miR-26a、miR-130a表达水平,蛋白免疫印迹法检测外周血单个核细胞中PI3K、Akt蛋白表达;采用Pearson相关性分析miR-26a、miR-130a表达水平与PI3K、Akt蛋白表达的相关性。患者治疗后行1个月随访,根据改良Rankin量表（mRS）评分分为预后良好组（n=55例,mRS评分≤2分）和预后不良组（n=43例,评分＞2分）。收集患者的一般资料,采用Logistic回归模型分析老年ACI患者预后的影响因素。结果：研究组血清miR-26a表达水平高于对照组,miR-130a表达水平低于对照组（P＜0.05）。研究组PI3K蛋白表达量低于对照组,Akt蛋白表达量高于对照组（P＜0.05）。Pearson相关性分析显示,miR-26a表达水平与PI3K蛋白表达量呈负相关、与Akt蛋白表达量呈正相关,miR-130a表达水平与PI3K蛋白表达量呈正相关、与Akt蛋白表达量呈负相关（P＜0.05）。预后良好组与预后不良组在年龄、房颤占比、入院时美国国立卫生研究院卒中量表（NIHSS）评分、血糖及血清miR-26a、miR-130a表达水平方面比较具有统计学意义（P＜0.05）。Logistic回归分析显示,年龄偏大、入院时NIHSS评分偏高、血糖偏高及血清miR-26a表达水平升高为老年ACI患者预后不良的危险因素,miR-130a表达水平升高为其保护因素（P＜0.05）。结论：老年ACI患者血清miR-26a表达水平升高、miR-130a表达水平下降,与PI3K/Akt信号通路密切相关,是老年ACI患者预后的影响因素。     

Induction by Fructose Force-Feeding of Histone H3 and H4 Acetylation at Their Lysine Residues around the Slc2a5 Gene and Its Expression in Mice

It has been reported that fructose force-feeding rapidly induced jejunal Slc2a5 gene expression in rodents. We demonstrate in this study that acetylation at lysine (K) 9 of histone H3 and acetylation at K5 and K16 of histone H4 were more enhanced in the promoter/enhancer to transcribed regions of the Slc2a5 gene in fructose force-fed mice than in glucose force-fed mice. However, fructose force-feeding did not induce acetylation at K14 of histone H3, or at K8 and K12 of histone H4 around the Slc2a5 gene. These results suggest that fructose force-feeding induced selective histone acetylation, particularly of H3 and H4, around the jejunal Slc2a5 gene in mice.     

施锌对石灰性褐土上小白菜光合作用及保护酶活性的影响

通过田间试验的方法,采用30(低锌)、75(中锌)和120kg/hm2ZnSO4(高锌)3个施锌水平,研究施锌对山西省石灰性褐土上小白菜叶片叶绿素含量、净光合速率、硝酸还原酶(NR)活性、抗氧化酶活性及丙二醛(MDA)含量的影响.结果表明:随着施锌量的提高,小白菜叶片各叶绿素组分含量、总叶绿素含量、净光合速率和NR活性均呈先升后降的变化趋势,并均以中锌处理最大,且中、高锌处理显著高于对照(不施锌);小白菜叶片过氧化物酶(POD)和过氧化氢酶(CAT)活性也呈先升后降的趋势,而超氧化物歧化酶(SOD)活性持续增加,CAT、POD和SOD活性分别在低锌、中锌和高锌处理时达到最大值,所有施锌处理的3种保护酶活性均显著高于缺锌对照;各处理小白菜叶片MDA含量随着施锌量增大而逐渐降低,且所有施锌处理均显著低于缺锌对照.研究发现,施锌能有效增强石灰性褐土上小白菜叶片光合能力和氮同化能力,显著提高其抗氧化酶系活性,并以75kg/hm2ZnSO4效果最佳.     

PI3K/AKT/mTOR信号通路及其在卵巢癌治疗中的应用

卵巢癌是女性生殖系统常见的恶性肿瘤,发病率居于妇科恶性肿瘤第三位,死亡率居于妇科恶性肿瘤之首。目前对卵巢癌的标准治疗包括肿瘤细胞减灭术及卡铂和紫杉醇的联合化疗。PI3K/AKT/mTOR信号通路在卵巢癌的细胞增殖、侵袭、细胞周期进展、血管生成及耐药中发挥重要作用,是卵巢癌中最常发生改变的细胞内途径。本文对PI3K/AKT/mTOR信号通路及其在卵巢癌增殖和进展中的影响、PI3K/AKT/mTOR信号通路抑制剂在卵巢癌中的治疗应用做简要综述。