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Glycosylphosphatidylinositol-specific phospholipase D1 (GPLD1) hydrolyzes inositol phosphate linkages in proteins anchored to the cell membrane. Mice overexpressing GPLD1 show enhanced neurogenesis and cognition. Snell dwarf (DW) and growth hormone receptor knockout (GKO) mice show delays in age-dependent cognitive decline. We hypothesized that augmented GPLD1 might contribute to retained cognitive function in these mice. We report that DW and GKO show higher GPLD1 levels in the liver and plasma. These mice also have elevated levels of hippocampal brain-derived neurotrophic factor (BDNF) and of doublecortin (DCX), suggesting a mechanism for maintenance of cognitive function at older ages. GPLD1 was not increased in the hippocampus of DW or GKO mice, suggesting that plasma GPLD1 increases elevated these brain proteins. Alteration of the liver and plasma GPLD1 was unaltered in mice with liver-specific GHR deletion, suggesting that the GH effect was not intrinsic to the liver. GPLD1 was also induced by caloric restriction and by each of four drugs that extend lifespan. The proteome of DW and GKO mice is molded by selective translation of mRNAs, involving cap-independent translation (CIT) of mRNAs marked by N

Altered adipose tissue may contribute to the longevity of Snell dwarf and growth hormone receptor (GHR) knock-out mice. We report here that white (WAT) and brown (BAT) fat have elevated UCP1 in both kinds of mice, and that adipocytes in WAT depots turn beige/brown. These imply increased thermogenesis and are expected to lead to improved glucose control. Both kinds of long-lived mice show lower levels of inflammatory M1 macrophages and higher levels of anti-inflammatory M2 macrophages in BAT and WAT, with correspondingly lower levels of TNFα, IL-6, and MCP1. Experiments with mice with tissue-specific disruption of GHR showed that these adipocyte and macrophage changes were not due to hepatic IGF1 production nor to direct GH effects on adipocytes, but instead reflect GH effects on muscle. Muscles deprived of GH signals, either globally (GKO) or in muscle only (MKO), produce higher levels of circulating irisin and its precursor FNDC5. The data thus suggest that the changes in adipose tissue differentiation and inflammatory status seen in long-lived mutant mice reflect interruption of GH-dependent irisin inhibition, with consequential effects on metabolism and thermogenesis.

In order to adapt to the low temperature environment, organisms transmit excitement to the central system through the thermal sensing system, which is a classic reflex reaction. The cold receptor GLR-3 perceives cold and pro duces cold avoidance behavior through peripheral sensory neurons ASER. In order to further understand the gene encoding of the cold sensing glr-3 gene and the evolution of its homologous gene group function and protein function, the nucleotide sequence and amino acid sequence of the glr-3 gene and its homologous gene in 24 species were obtained and compared. By clustering with the GRIK2 gene sequence of Rana chensinensis, the bio informatics method was used to predict and sequence analyze the change of gene, evolution rate, physical and chemical properties of protein, glycosyla tion sites, phosphorylation sites, secondary structure and tertiary structure of protein. The analysis results show that the glr-3 gene and its homologous gene have obvious positive selection effect. The protein prediction analysis showed that the glr-3 gene and its homologous genes encoded proteins in these 25 species were hydrophilic proteins, and the proportion of side chains of aliphatic amino acids was high. The transmembrane helix was widespread and there were more N-glycosylation sites and O-glycosylation sites. The protein phosphorylation sites encoded were serine, threonine and tyrosine phosphorylation sites. Secondary structure prediction showed that the secondary structure units of the encoded protein were α-helix, β-turn, random coil and extended chain, and the proportion of α-helix was the larg est. This study provides useful information on the evolution and function of the cold sensing gene glr-3 and its homologous genes.

To investigate the effect of miR-223 on thyroid cancer cells, further to study its potential mechanisms. The difference in miR-223 expression between normal thyroid Nthy-ori3-l cells and thyroid cancer SW579 cells was detected by PCR. The miR-223 overexpression and silencing vector transfection were verified by qRT-PCR. To further investigate the role of miR-223 in AQP-1, the AQP-1 siRNA vector was transfected on the basis of transfection of miR-223 inhibitor vector. The cell proliferation was detected by plate cloning, MTT, and cellular immunofluorescence assays. Cell cycle and apoptosis were detected by flow cytometry. Western blot was used to detect the expression of AQP-1 protein. The expression of miR-223 in SW579 cells was higher than that in normal cells. After transfection with miR-223 mimic, miR-223 expression was increased in SW579 cells. MiR-223 inhibitor transfection can inhibit SW579 cells proliferation, promote apoptosis, and inhibit cell cycle G0/G1 arrest. The SW579 cells proliferation was decreased, and the apoptosis rate was increased after transfection of AQP-1 silencing vector. Compared with the AQP-1 siRNA group, the SW579 cells proliferation rate was further reduced, and the apoptosis rate was significantly increased after co-transfection of miR-223 silencing vector and AQP-1 silencing vector. AQP-1 protein was highly expressed in SW579 cells, and miR-223 inhibitor can down-regulate the expression of APQ-1 protein. The expression AQP-1 protein was significantly reduced after transfected with AQP-1 silencing vector. Inhibition of miR-223 expression could suppress proliferation and promote apoptosis of SW579, and its mechanism is related to down-regulation of APQ-1 protein expression.

Cadmium (Cd) accumulates with aging and is elevated in long-lived species. Metallothioneins (MTs), small cysteine-rich proteins involved in metal homeostasis and Cd detoxification, are known to be related to longevity. However, the relationship between Cd accumulation, the role of MTs, and aging is currently unclear. Specifically, we do not know if long-lived species evolved an efficient metal stress response by upregulating their MT levels to reduce the toxic effects of environmental pollutants, such as Cd, that accumulate over their longer life span. It is also unknown if the number of MT genes, their expression, or both protect the organisms from potentially damaging effects during aging. To address these questions, we reanalyzed several cross-species studies and obtained data on MT expression and Cd accumulation in long-lived mouse models. We confirmed a relationship between species maximum life span in captive mammals and their Cd content in liver and kidney. We found that although the number of MT genes does not affect longevity, gene expression and protein amount of specific MT paralogs are strongly related to life span in mammals. MT expression rather than gene number may influence the high Cd levels and longevity of some species. In support of this, we found that overexpression of MT-1 accelerated Cd accumulation in mice and that tissue Cd was higher in long-lived mouse strains with high MT expression. We conclude that long-lived species have evolved a more efficient stress response by upregulating the expression of MT genes in presence of Cd, which contributes to elevated tissue Cd levels. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-021-00393-3.

Melanotic schwannoma (MS), a slowly growing nerve sheath tumor, is not a purely benign tumor. MS accounts for less than 1% of all nerve sheath tumors. We herein describe a rare case of MS and present a literature review focusing on the treatment of this disease. Twelve years before presentation at our hospital, a 41-year-old woman was examined because of an 8-month history of neck pain and 6-month history of upper extremity numbness and weakness. She underwent surgery to remove a tumor, and the pathological examination confirmed a diagnosis of MS. Twelve years later, at 53 years of age, the patient presented to our hospital with a 2-year history of neck pain and upper extremity numbness and weakness. Posterior cervical tumor resection was performed along with posterior cervical laminectomy, decompression and intraspinal space-occupying internal fixation, and radiotherapy. MS recurrence was confirmed. No tumor recurrence or metastasis was found after 7 months of follow-up. Recurrence of MS is rare, and its diagnosis depends on pathological features. Radical excision is the primary treatment for MS. Incomplete resection of MS is a risk factor for postoperative recurrence and metastasis. Furthermore, postoperative adjuvant radiotherapy should be performed to prevent recurrence and metastasis of MS.

Biological aging is the dominant risk factor for most chronic diseases. Development of anti-aging interventions offers the promise of preventing many such illnesses simultaneously. Cellular stress resistance is an evolutionarily conserved feature of longevity. Here, we identify compounds that induced resistance to the superoxide generator paraquat (PQ), the heavy metal cadmium (Cd), and the DNA alkylator methyl methanesulfonate (MMS). Some rescue compounds conferred resistance to a single stressor, while others provoked multiplex resistance. Induction of stress resistance in fibroblasts was predictive of longevity extension in a published large-scale longevity screen in C. elegans. Transcriptomic analysis implicated Nrf2 signaling in stress resistance provided by two protective compounds, cardamonin and AEG 3482. Molecules that conferred stress resistance also induced cellular inflammatory pathways, and other core pathways such as AMPK signaling. Small molecules identified in this work may represent attractive candidates to evaluate for their potential pro-health and pro-longevity effects in mammals.

Summary Studies of the mTOR pathway have prompted speculation that diminished mTOR complex-1 (mTORC1) function may be involved in controlling the aging process. Our previous studies have shown diminished mTORC1 activity in tissues of three long-lived mutant mice: Snell dwarf mice, growth hormone receptor gene disrupted mice (GHRKO), and in this article, mice deficient in the pregnancy-associated protein-A (PAPPA-KO). The ways in which lower mTOR signals slow aging and age-related diseases are, however, not well characterized. Here, we show that Snell, GHKRO, and PAPPA-KO mice express high levels of two proteins involved in DNA repair, O-6-methylguanine-DNA methyltransferase (MGMT) and N-myc downstream-regulated gene 1 (NDRG1). Furthermore, we report that lowering mTOR enhances MGMT and NDRG1 protein expression via post-transcriptional mechanisms. We show that the CCR4-NOT complex, a post-transcriptional regulator of gene expression, is downstream of the mTORC1 pathway and may be responsible for the upregulation of MGMT and NDRG1 in all three varieties of long-lived mice. Our data thus suggest a novel link between DNA repair and mTOR signaling via post-transcriptional regulation involving specific alteration in the CCR4-NOT complex, whose modulation could control multiple aspects of the aging process.

ATF4, a DNA-binding factor that modulates responses to amino acid availability and ribosomal function, has been shown to be altered in both liver and fibroblasts from two strains of long-lived mice, i.e. Snell dwarf and PAPP-A knockout mice. New data now show elevated ATF4 levels, and elevation of ATF4-dependent proteins and mRNAs, in liver of mice treated with acarbose or rapamycin, calorically restricted mice, methionine-restricted mice, and mice subjected to litter crowding. Elevation of ATF4, at least in liver, thus seems to be a shared feature of diets, drugs, genes, and developmental alterations that extend maximum lifespan in mice.

Skin and mucous membranes come in contact with external environment and protect tissues from infections by producing antimicrobial peptides. We report that human peptidoglycan recognition proteins 3 and 4 (PGLYRP3 and PGLYRP4) are secreted as 89-115-kDa disulfide-linked homo- and heterodimers and are bactericidal against several pathogenic and nonpathogenic transient, but not normal flora, Gram-positive bacteria. PGLYRP3 and PGLYRP4 are also bacteriostatic toward all other tested bacteria, which include Gram-negative bacteria and normal flora Gram-positive bacteria. PGLYRP3 and PGLYRP4 are also active in vivo and protect mice against experimental lung infection. In contrast to antimicrobial peptides, PGLYRPs kill bacteria by interacting with their cell wall peptidoglycan, rather than permeabilizing their membranes. PGLYRP3 and PGLYRP4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus, stomach, and intestine. Thus, we have identified the function of mammalian PGLYRP3 and PGLYRP4, and show that they are a new class of bactericidal and bacteriostatic proteins that have different structures, mechanism of actions, and expression patterns than antimicrobial peptides.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.

Rapamycin, an inhibitor of mTOR kinase, increased median lifespan of genetically heterogeneous mice by 23% (males) to 26% (females) when tested at a dose threefold higher than that used in our previous studies; maximal longevity was also increased in both sexes. Rapamycin increased lifespan more in females than in males at each dose evaluated, perhaps reflecting sexual dimorphism in blood levels of this drug. Some of the endocrine and metabolic changes seen in diet-restricted mice are not seen in mice exposed to rapamycin, and the pattern of expression of hepatic genes involved in xenobiotic metabolism is also quite distinct in rapamycin-treated and diet-restricted mice, suggesting that these two interventions for extending mouse lifespan differ in many respects.

Recent studies in our laboratory indicated that arsenite at 20μM significantly induces the apoptosis of HUVECs. In this study we analyzed miRNAs expression profiles upon arsenic exposure of these cells to explore the molecular mechanisms of arsenic-induced vascular toxicity. The expression of miRNAs was examined by Exiqon miRCURY™ LNA microRNA chips. We found that 85 miRNAs were up-regulated and 52 were down-regulated by arsenic treatment as compared to the control group. The expression of altered miRNAs was validated by quantitative reverse-transcription PCR (qRT-PCR). A number of DNA motifs were identified in the promoters of the perturbed miRNAs by promoter analysis using MEME software. Analysis of cellular functions by using DAVID Bioinformatics Resources revealed that phosphoproteins and genes involved in alternative splicing are among the top categories targeted by both up- and down-regulated miRNAs. In conclusion, the results show that the alteration of miRNAs expression might play crucial roles in arsenic-induced vascular injury.

SummaryPeptidoglycan recognition proteins (PGRPs) are structurally conserved from insects to mammals. Insect PGRPs have diverse host-defense functions. Mammalian PGRPs PGLYRP-1, PGLYRP-3, and PGLYRP-4 have bactericidal activity, while PGLYRP-2 has amidase activity. To extend the known functions of mammalian PGRPs, we examined whether they have immunomodulating activities in peptidoglycan-induced arthritis in mice. We demonstrate that PGLYRP-2 and Nod2 are both required for arthritis in this model. The sequence of events in peptidoglycan-induced arthritis is activation of Nod2, local expression of PGLYRP-2, chemokine production, and recruitment of neutrophils into the limbs, which induces acute arthritis. Only PGLYRP-2 among the four mammalian PGRPs displays this proinflammatory function, and PGLYRP-1 is anti-inflammatory. Toll-like receptor 4 (TLR4) and MyD88 are required for maturation of neutrophils before peptidoglycan challenge. Our results demonstrate that PGRPs, Nod2, and TLR4, representing three different types of pattern-recognition molecules, play interdependent in vivo roles in local inflammation.

Peptidoglycan recognition proteins (PGRPs) are structurally conserved through evolution, but their functions in innate immunity are different in invertebrates and vertebrates. We asked what the functions of PGRPs in fish are and whether they are indispensable for defense against infection, because fish are the first vertebrates that developed adaptive immunity, but still rely solely on innate immunity during early development of embryos. We identified and cloned three zebrafish PGRPs, and showed that they are highly expressed in eggs, developing embryos, and in adult tissues that contact external environment. Zebrafish PGRPs have both peptidoglycan-lytic amidase activity and broad-spectrum bactericidal activity, which is a unique feature. Furthermore, we demonstrated that in the developing zebrafish embryo one of these PGRPs is essential for defense and survival during bacterial infections. This is the first demonstration of an absolute requirement for innate immunity in defense against infections in fish embryos and for a PGRP protein for survival in vertebrates.

Human peptidoglycan recognition protein 2 (PGLYRP2) is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and is differentially expressed in the two major organs in the human body, liver and skin. PGLYRP2 has a high constitutive expression in the liver but is not expressed in healthy human skin. PGLYRP2 mRNA is also not expressed in cultured human keratinocytes but is highly induced upon exposure to bacteria. In this study we identified the transcription start site for pglyrp2 and demonstrated that the differential expression of PGLYRP2 in hepatocytes and keratinocytes is regulated by different transcription factors whose binding sequences are located in different regions of the pglyrp2 promoter. Induction of pglyrp2 in keratinocytes is regulated by sequences in the distal region of the promoter and requires transcription factors NF-kappaB and Sp1, whereas constitutive expression of pglyrp2 in a hepatocyte cell line is regulated by sequences in the proximal region of the promoter and requires transcription factors c-Jun and ATF2. Regulation of constitutive and inducible expression of pglyrp2 is important for systemic and local innate immune responses to bacterial infections.