MT1 Expression Research Articles

Zinc Homeostasis Alters Zinc Transporter Protein Expression in Vascular Endothelial and Smooth Muscle Cells.

Zinc is an important essential micronutrient with anti-oxidative and anti-inflammatory properties in humans. The role of zinc in signalling has been characterized in the nervous, endocrine, gastrointestinal, renal and reproductive systems. Relatively little is known regarding its role in the vascular system, but the role of zinc homeostasis in augmenting vascular health and vasorelaxation is emerging. Zinc transport proteins are integral to the protective function of zinc, but knowledge of their expression in vascular endothelial and smooth muscle cells is lacking. Human coronary artery endothelial cells and pulmonary artery smooth muscle cells were assessed for gene expression (RT-PCR) of SLC39A (ZIP), SLC30A (ZnT) and metallothionein (MT) families of Zn transporters and storage proteins. Protein expression (fluorescence confocal microscopy) was then analysed for the proteins of interest that changed mRNA expression: ZIP2, ZIP12, ZnT1, ZnT2 and MT1/2. Endothelial and smooth muscle cell mRNA expression of ZnT1, ZnT2 and MT1 was significantly downregulated by low and high Zn conditions, while ZIP2 and ZIP12 expression was induced by Zn depletion with the Zn chelator, TPEN. Changes in gene expression were consistent with protein expression levels for ZIP2, ZIP12 and MT1, where ZIP2 was localized to intracellular bodies and ZIP12 to lamellipodia. Vascular endothelial and smooth muscle cells actively regulate specific Zn transport and metallothionein gene and protein expressions to achieve Zn homeostasis.

Expression of Melatonin Receptor 1 in Rat Mesenteric Artery and Perivascular Adipose Tissue and Vasoactive Action of Melatonin

Melatonin is released by the pineal gland and can modulate cardiovascular system function via the G protein-coupled melatonin receptors MT1 and MT2. Most vessels are surrounded by perivascular adipose tissue (PVAT), which affects their contractility. The aim of our study was to evaluate mRNA and protein expression of MT1 and MT2 in the mesenteric artery (MA) and associated PVAT of male rats by RT-PCR and Western blot. Receptor localization was further studied by immunofluorescence microscopy. Effects of melatonin on neurogenic contractions were explored in isolated superior MA ex vivo by measurement of isometric contractile tension. MT1, but not MT2, was present in MA, and MT1 was localized mainly in vascular smooth muscle. Moreover, we proved the presence of MT1, but not MT2 receptors, in MA-associated PVAT. In isolated superior MA with intact PVAT, neuro-adrenergic contractile responses were significantly smaller when compared to arteries with removed PVAT. Pre-treatment with melatonin of PVAT-stripped arterial rings enhanced neurogenic contractions, while the potentiating effect of melatonin was not detected in preparations with preserved PVAT. We hypothesize that melatonin can stimulate the release of PVAT-derived relaxing factor(s) via MT1, which can override the direct pro-contractile effect of melatonin on vascular smooth muscle. Our results suggest that melatonin is involved in the control of vascular tone in a complex way, which is vessel specific and can reflect a sum of action on different layers of the vessel wall and surrounding PVAT.

5-Azacytidine upregulates melatonin MT1 receptor expression in rat C6 glioma cells: oncostatic implications.

The multiple physiological effects of the indoleamine melatonin, are mediated primarily by its two G protein-coupled MT1 and MT2 receptors. Treatment with histone deacetylase (HDAC) inhibitors, including valproic acid (VPA) and trichostatin A, upregulates melatonin receptors in cultured cells and the rat brain. VPA increases histone H3 acetylation of the MT1 gene promoter in rat C6 glioma cells, indicating that this epigenetic mechanism is involved in upregulation of MT1 expression. Since HDAC inhibitors can alter DNA methylation, the possible involvement of this other epigenetic mechanism, in the regulation of MT1 expression, was examined. RT-qPCR and western blotting studies confirmed that treatment with the DNA demethylating agent, 5-azacytidine (AZA; 10 or 20µM) for 24 or 48h, suppressed DNA methyltransferase 1 mRNA and protein expression in C6 cells. Subsequent treatment with AZA (1-25µM) for 24h, revealed a significant concentration-dependent upregulation of MT1 mRNA expression. Moreover, a combination of 5µM AZA plus 3mM VPA caused a synergistic upregulation of the MT1 receptor, which exceeded the sum of the independent effects of these drugs. These results show that DNA methylation plays a role in the regulation of the MT1 receptor, consistent with the established effects of this major epigenetic mechanism on gene transcription. Combinatorial epigenetic regulation of melatonin receptor expression could provide novel strategies for enhancing the oncostatic, neuroprotective and other therapeutic benefits of this pleiotropic indoleamine and its receptor agonists.

Zinc Deficiency Aggravation of ROS and Inflammatory Injury Leading to Renal Fibrosis in Mice.

Zinc (Zn) is a trace element with a variety of anti-inflammatory and antioxidant effects. Zn deficiency is related to tissue fibrosis. The present study was designed to investigate the effect of Zn on renal fibrosis. Mouse models were successfully established by feeding mice diets with different concentrations of Zn. Zn deficiency induced a decrease in Zn levels in kidney tissue. The results also revealed renal vasodilation, hyperemia, and inflammatory cell infiltration, and the levels of creatinine and urea nitrogen were increased. Furthermore, the TUNEL results showed a large degree of renal cell necrosis caused by Zn deficiency. Meanwhile, the corresponding antioxidant and anti-inflammatory regulators (MT-1, MT-2, Nrf2, and TGF-β1) were detected by RT-PCR, showing that the expression of MT-1, MT-2, and Nrf2 decreased but that TGF-β1 expression increased. The results of Sirius red staining proved that the expression of collagen was increased by Zn deficiency. The immunohistochemical experiments found that the expression of α-smooth muscle actin (α-SMA) increased. ELISA showed that the expression of Collagen I, III, and IV; fibronectin (FN); and inflammatory factors (TNF-α and IL-1β) were remarkably increased. The expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, and TIMP-1, which are extracellular matrix-regulating molecules, was detected by RT-PCR. The results showed that the expression of TIMPs was increased but that the expression of MMPs was decreased. We also obtained consistent results in vivo. All the experimental results indicated that Zn deficiency could aggravate fibrosis by increasing inflammation in the kidney.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

The immune defense response of Pacific white shrimp (Litopenaeus vannamei) to temperature fluctuation

Temperature is a significant environmental factor contributing to the success of aquaculture. To investigate the immune defense response during temperature fluctuation, Litopenaeus vannamei (L. vannamei) was treated under conditions of gradual cooling from an acclimation temperature of 28 °C (C group) to 13 °C (T group) in 2 days with a cooling rate of 7.5 °C/d and then rewarmed to 28 °C (R group) using the same rate. Relative expression of immune defense system-related genes and intestinal microbial composition in L. vannamei were investigated. The results showed that with a decrease in temperature, the expression of TLR, IMD, proPO and Casp3 in intestine was significantly decreased, while the expression of Muc-3A, Muc-5AC, Muc-17, IAP, p53, HSP70, MT and Fer was significantly increased after cooling. After temperature recovery, gene expression generally showed a trend of recovering to the normal level (C). Intestinal microbial analysis showed that, compared with the C group, the Chao and Ace indexes, the relative abundance of microflora from the Phylum Bacteroidetes, Class Alphaproteobacteria, and Class Bacteroidia, significantly decreased in the R group. The results revealed that cold-stress may decrease microbial community richness, alter the bacterial community in general and reduce shrimp immunity to pathogens and antibacterial activity. As a result, during temperature fluctuation shrimp may mobilize the immune defense system through upregulating the expression of Muc genes, anti-apoptosis related genes, and antioxidant related genes in order to maintain organism homeostasis as well as to repair damaged intestinal tissue.

Single‐cell RNA Sequencing reveals molecular heterogeneity of glia within mouse sympathetic ganglia

Stellate ganglion neurons, important mediators of cardiopulmonary neurotransmission, are surrounded by satellite glial cells (SGCs), which are essential for the maintenance and development of neurons. However, little is known about the heterogeneity and physiological functions of glia in adult sympathetic ganglia, and their role in modulating neurotransmission. Therefore, we performed single‐cell RNA sequencing of dissociated mouse stellate ganglia (n=8 animals), specifically focusing on the SGCs. SGCs were identified by high expression of glial specific transcripts, S100b and Fabp7 (n = 13370 cells). Microglia and Schwann cells were identified by expression of C1qa/C1qb/C1qc and Ncmap/Drp2, respectively, and excluded from further analysis. Subclustering of the SGCs revealed six distinct transcriptomic profiles. Expression levels of different glial marker genes were compared between these six subgroups to describe and study their distinct molecular profiles. The first subgroup was classified as glia‐progenitors (n = 1689; principle markers: Ng2 and Sox2). Pathway analysis demonstrated activation of pathways involved in self‐renewal of neural precursor cells and maintenance of radial glia cells. The second subgroup was identified as quiescent ‘astrocyte‐like’ glia (n = 3710; principle markers: Id3 and Aldoc). Accordingly, pathway analyses demonstrated active pathways involved in regulation of quantity and morphology of astrocytes. A third cluster (n = 2930) showed many similarities to the second cluster, but had a more reactive transcriptomic profile. Activated pathways included ones that were associated with astrocyte hypertrophy and quantity of reactive astrocytes. Hence, this cluster is referred to as the more active 'astrocyte‐like' cells and characterized by increased expression of the early response markers Mt1 and Klf2. The fourth cluster had a very distinct transcriptomic profile and had high expression levels of genes involved in the inflammatory response (n = 808; principle markers: Ifit3b and Gm4951). Correspondingly, pathways involved in immune response of cells and astrocytosis in the brain were enriched. Therefore, these cells may represent highly activated 'astrocyte‐like' SGCs. The fifth and sixth cluster were characterized by the expression of genes involved in axon development or maintenance and therefore showed more similarities with oligodendrocytes. These clusters were referred to as the 'oligodendrocyte‐like cells' and could be separated in myelinating ‘oligodendrocyte‐like’ cells (n = 1507; markers: Egr2 and Cryab) and non‐myelinating ‘oligodendrocyte‐like’ cells (n = 951; markers: Gap43 and Kcna1). Accordingly, both clusters showed much overlap in their activated pathways, most of which were associated with the cell cycle of oligodendrocytes or Schwann cells. However, pathways associated with cholesterol syntheses were downregulated in the sixth cluster. Our findings indicate transcriptomic heterogeneity within SGCs in mouse stellate ganglia and suggest functional subgroups that may subserve various physiological roles in health and disease.tSNE plot depicting the different subclusters of satellite glial cells within mouse sympathetic ganglia.Figure 1Cladogram depicting the relations between the different satellite glial cell subclusters. The different subclusters were characterised by their principle genetic markers.Figure 2

Genotoxicity and oxidative damage in zebrafish (Danio rerio) after exposure to effluent from ethyl alcohol industry

In this research, the toxicological effect of untreated wastewater from of ethyl alcohol industry was evaluated on the zebrafish (Danio rerio) under experimental conditions. Fish were treated with zero, half, one and two percent of sewage effluent for 21 days. Toxic effects were monitored in liver by determining biochemical indicators, oxidative stress biomarkers, and the expression of genes involved in the detoxification. Results showed that Sod1, Gstp-1a, Gpx1a gene expressions were significantly increased in the hepatocytes after 21 days at 2.0% sewage exposure. Sewage exposure also significantly increased Gsr, Ces2 and Cyp1a, Mt1 and Mt2 gene expression in the hepatocytes of zebrafish as compared to the reference group (P < 0.01). Total cellular antioxidants, malondialdehyde (MDA) levels, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) activities in fish exposed to 1 and 2% of sewage were significantly higher than the control group (P < 0.01), whereas alanine aminotransferase (ALT) was only increased in fish exposed to 2% sewage (P < 0.01). A significant decrease in gamma-glutamyl-transferase (GGT) activity in fish exposed to 2% effluent was found (P < 0.01). Catalase (CAT) activity was increased in zebrafish exposed to all concentrations of effluent. The transcriptional analysis of the detoxification-related genes and the changes in the biochemical indicators evidenced that drainage of sewage effluents from the ethyl alcohol company is a serious threat to the health of aquatic animals in the Khorram-Rood River. These results will contribute to further study on the impact of sewage effluents of the alcohol industry on aquatic organisms.

Melatonin concentration in peripheral blood and melatonin receptors (MT1 and MT2) in the testis and epididymis of male roe deer during active spermatogenesis

Melatonin regulates male reproductive function in seasonal and non-seasonal breeder mammals. The presence of melatonin membrane receptors (MT1 and MT2) in the testis and epididymis has been demonstrated in several species. Wild roe deer are a short-day breeding species characterised by a short rutting season lasting from mid-July to mid-August. The aim of this study was to determine the concentration of melatonin in the peripheral blood and the presence of MT1 and MT2 receptors in the testis and epididymis in male roe deer during the pre-rut (May), rut (July/August) and post-rut (September) periods. The melatonin concentration was higher in May (522.50 ± 54.20 pg/mL) compared to July/August (258.50 ± 36.82 pg/mL; P < 0.05). During September, the melatonin concentration was higher (393.50 ± 36.77 pg/mL) than in July/August (P < 0.05) but lower than in May (P < 0.05). Immunohistochemical analysis showed the presence of MT1 and MT2 receptors in Leydig cells, Sertoli cells and germ cells in the testis, in addition to the epithelial cells of the epididymis caput, corpus and cauda. MT1 and MT2 receptor expression in the testis and epididymis, assessed by Western blot, was higher in May and July/August (when spermatogenic and steroidogenic activity restarts and reaches its peak, respectively) compared to September (when spermatogenic and steroidogenic activity decreases). This could indicate a stimulatory effect of melatonin on testicular (i.e., steroidogenesis and spermatogenesis) and epididymal (i.e., spermatozoa maturation) function in male roe deer through the MT1 and MT2 receptors. Our results form the basis for further studies into the detailed mechanism of action of melatonin through MT1 and MT2 receptors for optimal reproductive activity in male roe deer and other mammals.

Polyvinyl chloride (PVC) plastic fragments release Pb additives that are bioavailable in zebrafish

Plastic polymers such as polyvinyl chloride (PVC) may contain chemical additives, such as lead (Pb), that are leachable in aqueous solution. The fragmentation into microplastics (MPs) of plastics such as PVC may facilitate desorption of chemical additives and increase exposure of aquatic animals. In this study, the role of chemical additives in the aqueous toxicity of PVC, high-density polyethylene (HDPE) and polyethylene terephthalate (PET) MPs were investigated in early-life stage zebrafish (Danio rerio) by assessment of changes in expression of biomarkers. Exposure of zebrafish larvae to PVC for 24 h increased expression of metallothionein 2 (mt2), a metal-binding protein, but no changes in expression of biomarkers of estrogenic (vtg1) or organic (cyp1a) contaminants were observed. HDPE and PET caused no changes in expression of any biomarkers. A filtered leachate of the PVC also caused a significant increase in expression of mt2 and indicated that a desorbed metal additive likely elicited the response in zebrafish. Metal release was confirmed by acid-washing the MPs which mitigated the response in mt2. Metal analysis showed Pb leached from PVC into water during exposures; at 500 mg PVC L−1 in water, 84.3 ± 8.7 μg Pb L−1 was measured after 24 h. Exposure to a Pb-salt at this concentration caused a comparable mt2 increase in zebrafish as observed in exposures to PVC. These data indicated that PVC MPs elicited a response in zebrafish but the effect was indirect and mediated through desorption of Pb from PVC into the exposure water. Data also indicated that PVC MPs may act as longer-term environmental reservoirs of Pb for exposure of aquatic animals; the Pb leached from PVC in 24 h in freshwater equated to 2.52% of total Pb in MPs leachable by the acid-wash. Studies of MPs should consider the potential role of chemical additives in toxicity observed.

Expression of melatonin receptors and CD4 in the ovine thymus, lymph node, spleen and liver during early pregnancy.

As a pineal gland hormone, melatonin acts through its receptors to modulate the immune system. The immune system is composed of primary and secondary organs, and immune organs are adapted to the presence of the fetal alloantigen during pregnancy. However, it is unclear whether melatonin affects maternal immune organs during early pregnancy in sheep. In this study, the ovine thymus, lymph node, spleen and liver were sampled at day 16 of the oestrous cycle, and at days 13, 16 and 25 of pregnancy. The expression of melatonin receptor 1A (MT1), melatonin receptor 1B (MT2) and cluster of differentiation 4 (CD4) was detected by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry experiments. Our results showed that during early pregnancy there was an upregulation of MT1 mRNA and protein in the thymus, lymph node and liver, and there was a downregulation in the spleen. The expression of MT2 mRNA and protein was increased in the thymus but decreased in the spleen and liver, and there was no significant change in the lymph node during early pregnancy. CD4 protein was upregulated in the thymus, lymph node and liver, but there were no significant changes in the spleen during early pregnancy. In conclusion, early pregnancy induces tissue-specific expression of MT1, MT2 and CD4, which may be due to the different functions of the thymus, lymph node, spleen and liver. Further, melatonin is involved in immune regulation of the maternal thymus, lymph node, spleen and liver during early pregnancy in sheep.

Farnesoid X receptor (FXR) activation induces the antioxidant protein metallothionein 1 expression in mouse liver

Farnesoid X receptor (FXR) is a metabolic nuclear receptor, which protects liver from many endogenous and exogenous injuries. Metallothioneins (MTs) belong to a low-molecular-weight protein family involved in metal homeostasis and the regulation of hepatic oxidative stress. In the present study, we aimed to investigate the effect of FXR on hepatic MT1 expression and the underlying mechanism. C57BL/6 mice or primary cultured mouse hepatocytes were treated with the synthetic FXR ligand GW4064 or natural ligand CDCA. RNA-Sequencing (RNA-seq) analysis was performed to identify gene expression profile in the livers of mice treated with GW4064. Real-time PCR and Western blot were applied to determine the expression of MT1 and other FXR target genes in the livers of mice and primary hepatocytes treated with GW4064 and CDCA. Cellular and subcellular locations of MT1 in the livers of mice treated with GW4064 were examined using immunohistochemistry assay. FXR small interfering RNAs (siRNA) was transfected to silence FXR. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were utilized to confirm the regulation of MT1 gene promoter activity by FXR. RNA-seq analysis revealed that GW4064 treatment significantly induced MT1 expression in mouse liver. Consistently, MT1 expression in the hepatocytes of mouse livers and cultured hepatocytes was upregulated by GW4064 as well as CDCA. In addition, adenovirus-mediated overexpression of FXR markedly increased, while siRNA-mediated FXR silencing significantly suppressed MT1 expression in cultured hepatocytes. Luciferase reporter and ChIP assays further confirmed that the MT1 gene was under the direct control of FXR. Collectively, our findings demonstrate that MT1 is a novel target gene of FXR and may contribute to antioxidative capacity of FXR in liver diseases.

The Molecular Anatomy of Mouse Skin during Hair Growth and Rest.

Skin homeostasis is orchestrated by dozens of cell types that together direct stem cell renewal, lineage commitment, and differentiation. Here, we use single-cell RNA sequencing and single-molecule RNA FISH to provide a systematic molecular atlas of full-thickness skin, determining gene expression profiles and spatial locations that define 56 cell types and states during hair growth and rest. These findings reveal how the outer root sheath (ORS) and inner hair follicle layers coordinate hair production. We found that the ORS is composed of two intermingling but transcriptionally distinct cell types with differing capacities for interactions with stromal cell types. Inner layer cells branch from transcriptionally uncommitted progenitors, and each lineage differentiation passes through an intermediate state. We also provide an online tool to explore this comprehensive skin cell atlas, including epithelial and stromal cells such as fibroblasts, vascular, and immune cells, to spur further discoveries in skin biology.

Disruption of Bone Zinc Metabolism during Postnatal Development of Rats after Early Life Exposure to Cadmium.

This current study was conducted to investigate whether bone tissue impairment induced by early life exposure to cadmium (Cd) during postnatal development could result from disruption to zinc (Zn) metabolism. For this reason, the offspring from mothers receiving either tap water, Cd, Zn or Cd + Zn during gestation and lactation periods were euthanized at PND21 and PND70. At the end of the lactation period (PND21), our results showed that exposure to Cd increased Cd accumulation and Zn depletion in the femur. Furthermore, calcium (Ca) level was reduced. At the molecular level, Cd induced an increase of MT-1 expression and caused an upregulation of ZIP2 accompanied with a down-regulation of ZnT5. Runx2, ALP, colα-1 and Oc mRNA levels were also decreased. In plasma, IGF-1 and osteocalcin concentrations were decreased. Further, Cd altered femoral growth by generating changes in the growth plate. Consequently, the toxic effect of Cd persisted at adult age (PND70) by decreasing bone volume (%BV/TV), bone mineral density (BMD) and Ca content and by increasing trabecular separation (Tb.Sp) in the distal femur. Interestingly, Zn supply provided total or partial corrections of several toxic effects of Cd. These data suggest that the increases of Zn bioavailability as well as the reduction of Cd accumulation in the femur following the changes in ZIP2 and ZnT5 expression are part of the mechanism involved in Zn protection against Cd toxicity on bone tissue.

Alteration of Zinc Transporter mRNA Expression in Zinc Depleted Condition by TPEN{N,N,Na2,Na2-Tetrakis(2-Pyridylmethyl)Ethylenediamine}: A Cell-Line Based Study.

Zinc deficiency is rapidly emerging as one of the important concerns in public health nutrition. Early diagnosis of zinc deficiency remains a major challenge. We investigated the expression level of different zinc transporters in zinc-deficient condition induced by TPEN, an intracellular zinc chelator in different cell lines like human monocyte (THP-1), skeletal muscle (RD), bone (Saos-2), liver (HepG2), representing different tissues which play key roles in zinc homeostasis. Cells were exposed to TPEN at various concentrations (2, 5, 10 μM) for 2 to12 h and mRNA levels of ZnT1 and MT were analyzed using qPCR. Statistical analysis was carried out using one-way ANOVA. ZnT1 expression was significantly different at 4 h with TPEN concentration of 2 μM and 5 μM as compared to untreated controls in THP-1, whereas in HepG2, significant differences were observed at 5 μM and 10 μM TPEN concentration after 6 h. In RD, significant differences were observed at 4 h in presence of 2 μM TPEN and in Saos2 expression was significantly different at 2 h with 2 μM, 5 μM, and 10 μM TPEN as compared to respective controls. Expression of MT in THP1 was significantly different at 2 h and 12 h control in presence of 2 μM, 5 μM and 10 μM TPEN, whereas in HepG2 significant differences were found at 2 μM, 5 μM, and 10 μM TPEN after 6 h of treatment. RD MT expression was significantly different in 10 μM for 12 h. Similarly, Saos2 expression was significantly different in the presence of 5 μM and 10 μM TPEN. Conclusions: This study may help in understanding the molecular cross talks among different zinc tissue storage depots during zinc deficiency and identification of early biomarkers for zinc deficiency.

Theophylline exerts complex anti-ageing and anti-cytotoxicity effects in human skin ex vivo.

Theophylline is a phosphodiesterase inhibitor that is being used clinically for asthma therapy. In addition, it is recognized as a cosmetic agent with possible anti-ageing and anti-oxidative properties. Nevertheless, how it affects human skin is still poorly examined. Theophylline (10 or 100µM) was administered to the culture medium of full-thickness human skin ex vivo for 24 or 72h. Theophylline stimulated protein expression of the anti-oxidant metallothionein-1 and mRNA levels of collagen I and III. Assessment of fibrillin-1 immunohistology revealed enhanced structural stability of dermal microfibrils. Theophylline also exerted extracellular matrix-protective effects by decreasing MMP-2 and MMP-9 mRNA levels, partially antagonizing the effects of menadione, the potent, toxic ROS donor. In addition, it decreased menadione-stimulated epidermal keratinocytes apoptosis. Interestingly, theophylline also increased the level of intracutaneously produced melatonin, that is the most potent ROS-protective and DNA damage repair neuromediator, and tendentially increased protein expression of MT1, the melatonin receptor. Theophylline also increased the expression of keratin 15, the stem cell marker, in the epidermal basal layer but did not change mitochondrial activity or epidermal pigmentation. This ex vivo pilot study in human skin shows that theophylline possesses several interesting complex skin-protective properties. It encourages further examination of theophylline as a topical candidate for anti-ageing treatment.

The capability of coenzyme Q10 to enhance heat tolerance in male rabbits: evidence from improved semen quality factor (SQF), testicular oxidative defense, and expression of testicular melatonin receptor MT1

Heat stress (HS) has a great influence on the etiology of male infertility. Coenzyme Q10 (CoQ10), known to have powerful antioxidant effects, has been reported to have such actions that are effective to treat infertility caused by HS. The aim of the present study was to investigate the antioxidative effect of CoQ10 on sperm quality, testicular antioxidant activities, and male fertility under HS. For this purpose, 18 mature male rabbits (aged 22 wk) of the Sinai Gabali breed were equally divided into 3 groups and placed at temperature-humidity index of 29 for 8 wk at a farm. The supplementation of CoQ10 at 0, 10, and 20 mg/kg of body weight was done in the first, second, and third groups, respectively. The results showed that the supplementation of CoQ10 had significant (P < 0.05) effect on semen quality factor (SQF) and testicular antioxidant activities by the supplementation of CoQ10. Moreover, a significant improvement in the concentration of testosterone, integrity of testicular DNA, and the expression of melatonin receptors was also observed, which were consistent with a significant improvement in buck fertility. The prolificacy was significantly increased (P < 0.05) in females when inseminated from bucks that were treated with CoQ10. Our results suggest that CoQ10 tends to decrease oxidative stress by enhancing testicular antioxidant activities, which are considered the most important factors for a buck's fertility. Hence, CoQ10 could be a suitable feed supplement to increase fertility, through enhancing the semen quality, in male rabbits and reducing the harmful effects of HS.

Expression and prognostic significance of melatonin receptor MT1 in patients with gastric adenocarcinoma.

Melatonin receptor type 1 (MTNR1A or MT1) is known to play an important role in cancer progression; however, its prognostic value for resected gastric adenocarcinoma (RGA) is unknown. In this study, we examined the potential of MT1 as a prognostic biomarker for RGA. The expression of the MT1 was evaluated in 67 patients with RGA by immunohistochemistry, and the relationship between MT1 levels and RGA prognosis was analyzed by Chi-square test, multivariate Cox regression, Kaplan-Meier method, and log-rank test. High MT1 expression was associated with a poor survival rate (29.0%, p=0.002) and the occurrence of metastasis (62.9%, p=0.004). Kaplan-Meier survival analysis and log rank tests revealed that patients with high expression of the MT1 had significantly shorter median overall survival compared to those with low expression (33.0 vs. 65.0 months, respectively; p=0.02). Multivariate Cox analysis indicated that the calculated death risk (hazard ratio [HR]) in patients with high expression levels of the MT1 increased to 2.68 (95% confidence interval [CI] 1.21-5.94, p=0.015), which was higher compared to those with low levels. HR of death was also high in patients with advanced T stage (2.51; 95 % CI 1.00-6.26, p=0.049) and metastasis (5.02; 95% CI 1.94-13.03, p=0.001). Our results showed that high MT1 expression in primary gastric adenocarcinoma tissues was associated with the occurrence of metastasis and poor prognosis. It may have prognostic significance as a potential biomarker in patients with RGA.

Forgotten partners and function regulators of inducible metallothioneins.

Metallothioneins are peculiar cysteine rich, heat resistant, small cellular plasma proteins expressed through almost all life forms. The currently established biological functions of metallothioneins are the homeostasis of essential metals and protection against toxic transitional metals (TM) alongside defence from oxidative stress by direct scavenging of reactive oxygen and nitrogen species (ROS and RNS). In mammals, among the four main evolutionary conserved forms, only the ubiquitously expressed metallothionein 1 and 2 (here abbreviated as MT) are inducible by TM, oxidative stress, glucocorticoids and starvation among various other stimuli. However, more than sixty years after being discovered, metallothioneins still bear unresolved issues about their possible physiological function and regulation. The biological function of MTs has still not been associated with the in vitro-demonstrated capacity of MT interaction with cellular molecules glutathione (GSH) or adenosine triphosphate (ATP), or with the possibility of direct iron-MT binding in the reducing intracellular environment of some organelles, e.g. lysosomes. Iron as the most abundant cellular TM is also one of the main physiological sources of ROS. Moreover, iron exhibits strain, sex and age differences that reflected ROS generation and MT induction in (patho)physiology and toxicology studies. A recent study showed that iron sex differences follows expression of both ferritin and MT leading to wide implications from essential TM interconnectivity to aging. This review places emphasis on biochemically proven but physiologically ignored interactions of MT with iron to stimulate advanced research for establishing a wide frame of the biological roles of MTs important for health and longevity.

Agomelatine treatment corrects impaired sleep-wake cycle and sleep architecture and increases MT1 receptor as well as BDNF expression in the hippocampus during the subjective light phase of rats exposed to chronic constant light.

Exposure to chronic constant light (CCL) has a detrimental impact on circadian rhythms of motor activity and sleep/wake cycles. Agomelatine is an atypical antidepressant showing a chronotropic activity. In this study, we explored the role of melatonin (MT) receptors and brain-derived neurotrophic factor (BDNF) in the brain in the mechanism underlying the effects of agomelatine on diurnal variations of motor activity, sleep/wake cycle, and sleep architecture in a rat model of CCL. In Experiment #1, home cage activity was monitored automatically with cameras for a period of 24h. The diurnal rhythm of MT1, MT2 receptors, and BDNF expression in the hippocampus and frontal cortex (FC), was tested using the ELISA test. In Experiment #2, rats were equipped with electroencephalographic (EEG) and electromyographic (EMG) electrodes and recordings were made under basal conditions (12:12 LD cycle + vehicle), LL + vehicle and LL + agomelatine (40mg/kg/day for 21days). The rats exposed to CCL showed an impaired diurnal rhythm of motor activity and sleep/wake cycle with reduced NREM sleep and delta power and increased REM sleep and theta power. The duration and number of episodes of the wake were diminished during the subjective dark phase in this group. The circadian rhythm of MT1 and MT2 receptors and their expression did not change in the hippocampus and FC under CCL exposure, while the BDNF levels in the hippocampus decreased during the subjective light phase. Agomelatine restored the diurnal rhythm of motor activity, disturbed sleep/wake cycle, and sleep architecture, which effect was accompanied by an increase in MT1 receptor and BDNF expression in the hippocampus at 10:00 in CCL rats. These findings support the value of agomelatine as an antidepressant that can adjust circadian homeostasis of motor activity and sleep/wake cycle in a CCL model.