PC2 Levels Research Articles

Different inflammatory signatures based on CSF biomarkers relate to preserved or diminished brain structure and cognition

Neuroinflammation is a hallmark of Alzheimer’s disease (AD) and both positive and negative associations of individual inflammation-related markers with brain structure and cognitive function have been described. We aimed to identify inflammatory signatures of CSF immune-related markers that relate to changes of brain structure and cognition across the clinical spectrum ranging from normal aging to AD. A panel of 16 inflammatory markers, Aβ42/40 and p-tau181 were measured in CSF at baseline in the DZNE DELCODE cohort (n = 295); a longitudinal observational study focusing on at-risk stages of AD. Volumetric maps of gray and white matter (GM/WM; n = 261) and white matter hyperintensities (WMHs, n = 249) were derived from baseline MRIs. Cognitive decline (n = 204) and the rate of change in GM volume was measured in subjects with at least 3 visits (n = 175). A principal component analysis on the CSF markers revealed four inflammatory components (PCs). Of these, the first component PC1 (highly loading on sTyro3, sAXL, sTREM2, YKL-40, and C1q) was associated with older age and higher p-tau levels, but with less pathological Aβ when controlling for p-tau. PC2 (highly loading on CRP, IL-18, complement factor F/H and C4) was related to male gender, higher body mass index and greater vascular risk. PC1 levels, adjusted for AD markers, were related to higher GM and WM volumes, less WMHs, better baseline memory, and to slower atrophy rates in AD-related areas and less cognitive decline. In contrast, PC2 related to less GM and WM volumes and worse memory at baseline. Similar inflammatory signatures and associations were identified in the independent F.ACE cohort. Our data suggest that there are beneficial and detrimental signatures of inflammatory CSF biomarkers. While higher levels of TAM receptors (sTyro/sAXL) or sTREM2 might reflect a protective glia response to degeneration related to phagocytic clearance, other markers might rather reflect proinflammatory states that have detrimental impact on brain integrity.

The G209R mutant mouse as a model for human PCSK1 polyendocrinopathy.

PCSK1 encodes an enzyme required for prohormone maturation into bioactive peptides. A striking number of SNPs and rare mutations in PCSK1 are associated with a range of clinical phenotypes. Infants bearing two copies of a catalytically inactivating mutation, such as G209R, exhibit life-threatening chronic diarrhea and subsequently develop systemic endocrinopathies. Using CRISPR/Cas9 technology, we have engineered a mouse model bearing a G209R missense mutation in exon 6 of the murine Pcsk1 locus. Most pups homozygous for the G209R mutation succumbed by day 2, and surviving pups were severely dwarfed. In homozygous (but not heterozygous) pups, blood glucose levels were significantly lower, accompanied by elevated plasma insulin-like immunoreactivity and accumulation of large quantities of unprocessed proinsulin in the pancreas. Peptide hormone processing was also aberrant in G209R mouse pituitary, with mature ACTH levels markedly reduced in homozygotes, accompanied by a significant accumulation of POMC. We also observed a significant reduction in PC1/3 protein in the brains of G209R homozygous mice by Western blotting, while PC2 levels remained unaffected. Most likely due to the continued presence of PC2, pituitary and brain levels of α-MSH were not impaired. Analysis of intestinal cell types indicated a modest reduction of enteroendocrine cells in G209R homozygotes. We suggest that the G209R Pcsk1 mouse model recapitulates many of the dramatic neonatal deficiencies of human patients with this homozygous mutation.

Aqueous Extracts of Pleurotus ostreatus and Hericium erinaceus Protect against Ultraviolet A-Induced Damage in Human Dermal Fibroblasts.

Pleurotus ostreatus (PO) and Hericium erinaceus (HE) have been traditionally used to treat various diseases, owing to their antioxidant, antimicrobial, neuroprotective, and antitumor effects. However, few studies have been reported on their antiaging effects. In this study, the antioxidant and antiaging activities of PO and HE aqueous extracts were investigated in ultraviolet A (UVA)-induced human dermal fibroblast cells (HDFs). The antioxidant properties of PO and HE aqueous extracts were measured by total polyphenol and ergothioneine content, and their antioxidant activity was analyzed with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical-scavenging assays. To demonstrate the antiaging effect of PO and HE aqueous extracts in UVA-induced HDFs, the secretion and mRNA expression of matrix metalloproteinase-1 (MMP-1), procollagen type I (PC1), and elastase were assessed by enzyme-linked immunosorbent assay (ELISA) and real-time PCR, respectively. The total polyphenol content in each extract was 13.6 and 11.7 mg gallic acid equivalents/g dry weight (DW), respectively, and the total ergothioneine content in each extract was 3.43 and 2.18 mg/g DW, respectively. The PO and HE extracts increased DPPH and ABTS radical-scavenging activity in a dose-dependent manner. In UVA-damaged HDFs, the extracts increased PC1 production but decreased MMP-1 production and elastase-1 activity. Furthermore, the mRNA levels of PC1, MMP-1, and elastase were recovered in the PO- and HE-treated UVA-irradiated HDFs compared to those in the irradiated control group. PO and HE aqueous extracts may be potentially used as a promising antiphotoaging agent.

Single and mixed effects of prenatal exposure to multiple bisphenols on hemoglobin levels and the risk of anemia in pregnant women

IntroductionBisphenols have endocrine-disrupting effects, which may disrupt hemoglobin (Hb) homeostasis and lead to anemia. However, the effects of bisphenols on anemia remain unknown. Therefore, we assessed the effects of single− and multiple−exposure to bisphenols on Hb levels and anemia of pregnant women. MethodsThe study involved 2035 pregnant women from Guangxi Zhuang Birth Cohort in China. Generalized linear regression, principal component analysis (PCA), quantile g-computation (Qgcomp), and Bayesian kernel machine regression (BKMR) were performed to examine the effects of serum bisphenols on Hb levels and the risk of anemia. ResultsAfter adjustment, elevated bisphenol A (BPA) levels were correlated with decreased Hb concentrations (β = −0.51; 95%CI: −0.92, −0.10) in the first trimester, and these correlations were more sensitive in mothers of males. Compared with the low-exposure group, bisphenol B (BPB) levels in the high-exposure group led to a 1.52 g/L (95%CI: −3.01, −0.03) decrease in Hb levels in the second trimester; tetrabromobisphenol A (TBBPA) levels in the high-exposure group led to a higher the risk of anemia in the third trimester (OR = 1.46; 95%CI: 1.07, 1.99); bisphenol F (BPF) in the high-exposure group led to lower Hb levels (β = −2.42; 95%CI:−4.69, −0.14) in mothers of male fetuses in the third trimester. Qgcomp showed that elevated levels of bisphenol mixture was correlated with (β = −1.42; 95%CI: −2.61, −0.24) decrease in Hb levels in the second trimester. PCA revealed a negative association between PC2 and Hb levels in the first trimester (β = −0.89; 95%CI: −1.61, −0.17). Similarly, a negative relationship was observed between PC1 and Hb levels in the third trimester among mothers with male fetuses (β = −1.00; 95%CI: −1.94, −0.06). ConclusionsPrenatal exposure to single and mixed bisphenols may decrease Hb levels and increase the risk of anemia during pregnancy, the associations may be greater in mothers with male fetuses than those with female fetuses.

Identification of Plasma Cell Antigen PC-1 as a Novel Cell Surface Marker of Germinal Center, Memory and Plasmacytoma B Cells.

The murine plasma cell (PC) alloantigen PC-1, also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), is expressed on PCs and by many non-lymphoid tissues. PC-1 is best known for its role in phosphate metabolism, hydrolyzing NTP to generate pyrophosphate. However, its expression and function in B lymphocytes remains unclear. Here we describe a previously unknown expression pattern of PC-1 in murine late stage B cells and B lymphomas. In normal mice, expression of PC-1, determined by flow cytometry, was very low on bone marrow pro-B, pre-B, immature B, and mature B cells, and on splenic follicular and marginal zone B cells. However, peritoneal B cells expressed significantly higher levels of PC-1 than splenic naïve B cells. CD5+ B1a cells expressed more PC-1 than B1b and B2 cells. Interestingly, PC-1 distinguishes two subsets of B1a: PC-1hi and PC-1lo. The PC-1hi B1a cells were IgMlo, B220lo, and larger in size while PC-1lo B1a cells were IgMhi, B220hi, and smaller in size. The PC-1hi B1a cells express higher levels of Blimp-1 transcripts than the PC-1lo B1a cells, indicating that the PC- 1hi B1a cells may exhibit features of pre-plasma cells. Furthermore, after immunization with NP-KLH, germinal center (GC) B cells, PCs and early memory B cells expressed 2–4-fold more PC-1 than naïve B cells. Naïve B cells stimulated with LPS or anti-IgM plus anti-CD40 antibodies also dramatically upregulated PC-1 expression. Finally, gene expression profiling studies of primary lymphomas in NFS.V(+) congenic mice revealed high expression levels of PC-1 in plasmacytomas, immunoblastic lymphomas, and centroblastic-follicular lymphoma types. These studies demonstrate that PC-1 can be a useful marker for identification of stages in GC and post-GC cell differentiation and of lymphomas with a GC or PC origin. The function of PC-1 in B cells is currently under investigation.

SUN-669 The G209R Mutant Mouse as a Model for Human PCSK1 Polyendocrinopathy

A striking number of SNPs and rare mutations have been identified in PCSK1, the gene that codes for the enzyme proprotein convertase 1/3 (PC1/3) which proteolytically activates prohormones within the secretory pathway. All infants bearing two copies of catalytically inactivating mutations, including G209R, exhibit severe neonatal malabsorption requiring parenteral nutrition for months and subsequently develop additional endocrinopathies, often including diabetes and obesity. In order to create a mouse model to explore the underlying mechanism of the malabsorption phenomenon and the endocrinopathies, a G209R point mutation was introduced into exon 6 of mouse Pcsk1 using CRISPR-Cas9 genome editing. Fifty-six live pups were collected at postnatal days one or two; however, most homozygous G209R mutant pups succumbed by day 2, and surviving pups were severely dwarfed. In homozygous, but not heterozygous pups, blood glucose levels were significantly lower with elevated plasma insulin-like immunoreactivity and accumulation of unprocessed proinsulin in G209R pancreas compared to the wild type pups from the same litters. The POMC product α-MSH (produced by PC2 from PC1/3-generated ACTH) has been strongly implicated in obesity mechanisms. We found pituitary POMC processing to ACTH was also affected by the G209R mutation in combined anterior and intermediate pituitary lobes. ACTH was markedly reduced in homozygote pituitary, with significant accumulation of POMC. Using Western blotting, we observed a significant reduction in PC1/3 protein in homozygote brains, while PC2 protein levels remained unaffected. Most likely due to the continued presence of PC2, pituitary and brain levels of α-MSH were not impaired, suggesting that α-MSH itself is not involved in the phenotype. Prior studies have shown that G209R PC1/3 is not efficiently trafficked out of the ER; further studies will examine the contribution of misfolded G209R PC1/3 to possible cellular ER stress, as well as determine peptide hormone levels in brain and peripheral tissues.

Prospective effects of perceived stress on immunosenescent principal components in older adults

Stress may drive aspects of immunological aging. The present investigation examined how perceived stress across five years predicts immunosenescence one year later, using principal component analysis as a dimension-reduction technique to characterize natural killer (NK) and T cell surface markers that independently contribute to variability in immunosenescence in older adults ( N = 140, 43% male, aged 60–92). Three principal components (PC) were identified. PC1 (49% variance explained) included indicators of NK cell exhaustion (NK56 dim : NKG2C+, FCR γ -, NKG2C + CD57+, NKG2C + FCR γ -, and FCR γ -CD57+). PC2 (23% variance explained) included indicators of T cell senescence (CD8+: CD28-, CD57+, and CD56+). PC3 (10% variance explained) included NK56dimCD57+. In a subset of older adults ( n = 68), perceived stress was measured biannually for up to five years preceding immune assessment. Person-specific perceived stress slopes and intercepts (centered at the last time point) were outputted and tested as main and interacting effects on PCs in regressions. There was a significant stress slope*intercept interaction predicting PC2 ( b = −53.5, SE = 18.85, p = 0.006). Older adults who decreased over time and ended low in perceived stress had the lowest PC2 levels ( b = 1.33, p = 0.004), whereas older adults who increased over time in stress had similar (high) PC2 levels whether or not they ended low/high in stress ( b = −0.81, p = 0.14). Ultimately, decreasing stress to low levels may retard age-associated aspects of T cell senescence and to a greater degree than NK cell aging in older adults.

Polycystin 2-dependent cardio-protective mechanisms revealed by cardiac stress.

Although autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple kidney cysts, the most frequent cause of death in ADPKD patients is cardiovascular disease. ADPKD is linked to mutations in PKD1 or pkd2, the genes that encode for the proteins polycystin 1 and polycystin 2 (PC1 and PC2, respectively). The cardiovascular complications have been assumed to be a consequence of renal hypertension and activation of renin/angiotensin/aldosterone (RAAS) pathway. However, the expression of PC1 and PC2 in cardiac tissue suggests additional direct effects of these proteins on cardiac function. We previously reported that zebrafish lacking PC2 develop heart failure, and that heterozygous Pkd2+/- mice are hypersensitive to acute β-adrenergic receptor (βAR) stimulation. Here, we investigate the effect of cardiac stress (prolonged continuous βAR stimulus) on Pkd2+/- mice. After βAR stimulation for 7days, wild-type (WT) mice had increased left ventricular mass and natriuretic peptide (ANP and BNP) mRNA levels. The WT mice also had upregulated levels of PC2 and chromogranin B (CGB, an upstream regulator of BNP). Conversely, Pkd2+/- mice had increased left ventricular mass, but natriuretic peptide and CGB expression levels remained constant. Reversal of the increased cardiac mass was observed in WT mice 3days after cessation of the βAR stimulation, but not in Pkd2+/- mice. We suggest that cardiac stress leads to upregulation of the PC2-CGB-BNP signaling axis, and this pathway regulates the production of cardio-protective natriuretic peptides. The lack of a PC2-dependent cardio-protective function may contribute to the severity of cardiac dysfunction in Pkd2+/- mice and in ADPKD patients.

Loss of prohormone convertase 2 promotes beta cell dysfunction in a rodent transplant model expressing human pro-islet amyloid polypeptide.

A contributor to beta cell failure in type 2 diabetes and islet transplants is amyloid formation by aggregation of the beta cell peptide, islet amyloid polypeptide (IAPP). Similar to the proinsulin processing pathway that generates insulin, IAPP is derived from a prohormone precursor, proIAPP, which requires cleavage by prohormone convertase (PC) 1/3 and PC2 in rodent pancreatic beta cells. We hypothesised that loss of PC2 would promote beta cell death and dysfunction in a rodent model of human beta cell proIAPP overexpression. We generated an islet transplant model wherein immune-deficient mouse models of diabetes received islets expressing amyloidogenic human proIAPP and lacking PC2, leading to restoration of normoglycaemia accompanied by increased secretion of human proIAPP. Blood glucose levels were analysed for up to 16weeks in transplant recipients and grafts were assessed for islet amyloid and beta cell number and death. Hyperglycaemia (blood glucose >16.9mmol/l) returned in 94% of recipients of islets expressing human proIAPP and lacking PC2, whereas recipients of islets that express human proIAPP and normal PC2 levels remained normoglycaemic for at least 16weeks. Islet graft failure was accompanied by a ∼20% reduction in insulin-positive cells, yet the degree of amyloid deposition and beta cell apoptosis was similar to those of controls expressing human proIAPP with functional PC2 levels. PC2 deficiency in transplanted mouse islets expressing human proIAPP promotes beta cell loss and graft failure. Our data suggest that impaired NH2-terminal processing and increased secretion of human proIAPP promote beta cell failure.

Exenatide substantially improves proinsulin conversion and cell survival that augment Ins2+/Akita beta cell function

Proinsulin folding imperfections cause extensive beta-cell defects known in diabetes. Here, we investigated whether exenatide can alleviate such defects in proinsulin conversion, beta-cell survival, and insulin secretion, in the Ins2+/Akita beta-cells that have a spontaneous mutation (Cys 96 Tyr) in the insulin 2 gene caused dominant negative misfolding problem. 15 or 120 min exenatide administration substantially improves glucose-stimulated insulin secretion, marked in the secreted insulin levels and proinsulin/insulin ratio. This improvement is mainly due to enhanced conversion of proinsulin to insulin, having nothing to do with the prohormone convertase PC1/3 and PC2 levels. The 15 min improvement is calcium-independent. The 120 min improvement is linked to calcium and/or cAMP dependent mechanisms. This efficacy is validated during longer treatment and in Akita islets. Exenatide improves Ins2+/Akita beta-cell survival and Akita mouse's glucose tolerance. The results suggest a potential of incretin mimetics in alleviating defective proinsulin conversion and other proinsulin misfolding consequences.

Posttranslational regulation of polycystin-2 protein expression as a novel mechanism of cholangiocyte reaction and repair from biliary damage.

Polycystin-2 (PC2 or TRPPC2), a member of the transient receptor potential channel family, is a nonselective calcium channel. Mutations in PC2 are associated with polycystic liver diseases. PC2-defective cholangiocytes show increased production of cyclic adenosine monophosphate, protein kinase A-dependent activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, hypoxia-inducible factor 1α (HIF-1α)-mediated vascular endothelial growth factor (VEGF) production, and stimulation of cyst growth and progression. Activation of the ERK/HIF-1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF-1α/VEGF pathway. PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2(-/-) knockout, bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment). Treatment of cholangiocytes with proinflammatory cytokines, nitric oxide donors, and endoplasmic reticulum stressors increased ERK1/2 phosphorylation, HIF-1α transcriptional activity, secretion of VEGF, and VEGF receptor type 2 phosphorylation and down-regulated PC2 protein expression without affecting PC2 gene expression. Expression of homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation, was increased. Pretreatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with nitric oxide donors or with endoplasmic reticulum stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of 3,5-diethoxycarbonyl-1,4-dihydrocollidine mice and of Mdr2(-/-) mice with the proteasome inhibitor bortezomib restored PC2 expression and significantly reduced the ductular reaction, fibrosis, and phosphorylated ERK1/2. In response to biliary damage, PC2 expression is modulated posttranslationally by the proteasome or the autophagy pathway, and PC2 down-regulation is associated with activation of ERK1/2 and an increase of HIF-1α-mediated VEGF secretion; treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases.

Regulation of prohormone convertase 2 protein expression via GPR40/FFA1 in the hypothalamus

Previous studies have shown that the administration of docosahexaenoic acid (DHA) or GW9508, a GPR40/FFA1 (free fatty acid receptor) agonist, facilitates β-endorphin release in the arcuate nucleus of the hypothalamus in mice. However, the mechanisms mediating β-endorphin release induced by GPR40/FFA1 agonists remain unknown. In this study, we focused on the changes in expression of hypothalamic prohormone convertase (PC) 2, which is a calcium-dependent subtilisin-related proteolytic enzyme. The intracerebroventricular injection of DHA or GW9508 significantly increased PC2 protein expression in the hypothalamus. This increase in PC2 expression was inhibited by pretreatment with GW1100, a GPR40/FFA1 antagonist. Furthermore, PC2 protein expression gradually increased over time after complete Freund's adjuvant. These increase in PC2 expression were inhibited by pretreatment with GW1100. However, GW1100 by itself had no effect on PC2 levels. Taken together, our findings suggest that activation of the hypothalamic GPR40/FFA1 signaling pathway may regulate β-endorphin release via PC2, and regulate the endogenous pain control system.

P1141 : Post-translational regulation of polycystin 2 (PC2) expression as a novel mechanism of cholangiocyte reaction to biliary damage and repair

Introduction: In polycystic liver diseases, defective PC2 leads to elevated production of cAMP, PKA-dependent activation of the ERK1/2 pathway, HIF1 -mediated VEGF production and stimulation of cyst growthand progression. Activation of the ERK/HIF1 /VEGF pathway in cholangiocytes is fundamental during repair from biliary damage. In this study we hypothesized that PC-2 levels can be modulated during biliary damage/repair. Results: PC2 protein expression was significantly reduced in livers from mice undergoing cholangiocyte damage (Mdr2-/--KO, BDL, treatment with dehydrocollidine–DDC). However, PC-2-gene expression was not reduced,suggesting that the decrease in PC2 was due to increaseddegradation. To understand if factors involved in biliary damage influence PC2 protein expression, mouse cholangiocytes were treated with pro-inflammatory cytokines, nitric oxide (NO) donors and ER stressors. Expression of PC2 protein was again significantly reduced, but not its gene expression. Noteworthy, downregulation of PC-2 leads to increased ERK1/2 phosphorylation, HIF1 transcription activity and secretion of VEGF. Expression of Herp and NEK, proteins that promote PC2 degradation via the proteasome complex was also increased. Pre-treatment with proteasome inhibitor restored the expression of PC2 in cells treated with cytokines but not in cells treated with NO donors or with ER stressors. In the latter conditions, PC2 degradation was inhibited by inhibition of PI3K, that blocks autophagosome andby blockade of lysosomes, suggesting a role for autophagy. Finally, treatment ofDDC-treatedmicewith the proteasome inhibitor bortezomib, significantly reduced the extent of the liver damage and restored PC2 expression. Conclusion: PC2 is modulated post-translationally by proinflammatory cytokines, ER-stressors and NO-donors and is reduced in mice with biliary damage. Down regulation of PC2 protein expression in cholangiocytes increases the ERK1/2/HIF1 /VEGFsecretion, thereby playing a pivotal role in the regulation of cholangiocyte response to biliary damage.Treatments able to restore PC2 expression may represent a new therapeutic approach in biliary diseases.

Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner.

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited nephropathy responsible for 4%-10% of end-stage renal disease cases. Mutations in the genes encoding polycystin-1 (PC1, PKD1) or polycystin-2 (PC2, PKD2) cause ADPKD, and PKD1 mutations are associated with more severe renal disease. PC1 has been shown to form a complex with PC2, and the severity of PKD1-mediated disease is associated with the level of the mature PC1 glycoform. Here, we demonstrated that PC1 and PC2 first interact in the ER before PC1 cleavage at the GPS/GAIN site and determined that PC2 acts as an essential chaperone for PC1 maturation and surface localization. The chaperone function of PC2 was dependent on the presence of the distal coiled-coil domain and was disrupted by pathogenic missense mutations. In Pkd2-/- mice, complete loss of PC2 prevented PC1 maturation. In Pkd2 heterozygotes, the 50% PC2 reduction resulted in a nonequimolar reduction (20%-25%) of the mature PC1 glycoform. Interbreeding between various Pkd1 and Pkd2 models revealed that animals with reduced levels of functional PC1 and PC2 in the kidney exhibited severe, rapidly progressive disease, illustrating the importance of complexing of these proteins for function. Our results indicate that PC2 regulates PC1 maturation; therefore, mature PC1 levels are a determinant of disease severity in PKD2 as well as PKD1.

Genetic associations with micronutrient levels identified in immune and gastrointestinal networks.

The discovery of vitamins and clarification of their role in preventing frank essential nutrient deficiencies occurred in the early 1900s. Much vitamin research has understandably focused on public health and the effects of single nutrients to alleviate acute conditions. The physiological processes for maintaining health, however, are complex systems that depend upon interactions between multiple nutrients, environmental factors, and genetic makeup. To analyze the relationship between these factors and nutritional health, data were obtained from an observational, community-based participatory research program of children and teens (age 6–14) enrolled in a summer day camp in the Delta region of Arkansas. Assessments of erythrocyte S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), plasma homocysteine (Hcy) and 6 organic micronutrients (retinol, 25-hydroxy vitamin D3, pyridoxal, thiamin, riboflavin, and vitamin E), and 1,129 plasma proteins were performed at 3 time points in each of 2 years. Genetic makeup was analyzed with 1 M SNP genotyping arrays, and nutrient status was assessed with 24-h dietary intake questionnaires. A pattern of metabolites (met_PC1) that included the ratio of erythrocyte SAM/SAH, Hcy, and 5 vitamins were identified by principal component analysis. Met_PC1 levels were significantly associated with (1) single-nucleotide polymorphisms, (2) levels of plasma proteins, and (3) multilocus genotypes coding for gastrointestinal and immune functions, as identified in a global network of metabolic/protein–protein interactions. Subsequent mining of data from curated pathway, network, and genome-wide association studies identified genetic and functional relationships that may be explained by gene–nutrient interactions. The systems nutrition strategy described here has thus associated a multivariate metabolite pattern in blood with genes involved in immune and gastrointestinal functions.Electronic supplementary materialThe online version of this article (doi:10.1007/s12263-014-0408-4) contains supplementary material, which is available to authorized users.

Phytochelatin–metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis

Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)-chelating peptides and by sequestering PC-metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As-PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC2 -Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC2 -Cd transport activity compared with PC2 -As. In contrast, barley vacuoles readily accumulated comparable levels of PC2 -Cd and PC2 -As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC-type transporters. Interestingly, barley vacuoles exhibited enhanced PC2 transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non-essential toxic metal(loid)s.

Induced pluripotent stem cells derived from patients with autosomal dominant PKD show reduced levels of PC2

Induced pluripotent stem cells derived from patients with autosomal dominant PKD show reduced levels of PC2

Ultraviolet B irradiation of the mouse eye induces pigmentation of the skin more strongly than does stress loading, by increasing the levels of prohormone convertase 2 and α-melanocyte-stimulating hormone

In previous studies, we made the unexpected finding that in mice, ultraviolet (UV)B irradiation of the eye increased the concentration of α-melanocyte-stimulating hormone (α-MSH) in plasma, and systemically stimulated epidermal melanocytes. To compare the extent of the pigmentation induced by social and restraint stress (which activate the hippocampus-pituitary system) with that induced by UVB irradiation. DBA/2 and sham-operated or hypophysectomized DBA/2 mice were subjected to local UVB exposure using a sunlamp directed at the eye, and two types of stress (social and restraint) were imposed. UVB irradiation of the eye or exposure to stress loading both increased the number of Dopa-positive melanocytes in the epidermis, and hypophysectomy strongly inhibited the UVB-induced and stress-induced stimulation of melanocytes. Irradiation of the eye caused a much greater increase in dopamine than did the stress load. Both UVB eye irradiation and stress increased the blood levels of α-MSH and adrenocorticotropic hormone (ACTH). In addition, the increase in plasma α-MSH was greater in animals subjected to UVB eye irradiation than in those subjected to stress loading, whereas the reverse occurred for plasma ACTH. UVB irradiation to the eye and stress loading increased the expression of prohormone convertase (PC)1/3 and PC2 in the pituitary gland. The increase in expression of pituitary PC2 was greater in animals subjected to UVB eye irradiation than to stress, whereas no difference was seen between the two groups for the increase in PC1/3. UVB eye irradiation exerts a stronger effect on pigmentation than stress loading, and is related to increased levels of α-MSH and PC2.

236 - Polycystins 1 and 2 as Novel Prognostic Markers in Colorectal Cancer

ABSTRACT Background/purpose Polycystins 1 and 2 (PC-1 and PC-2) constitute a family of cellular proteins detected in a variety of epithelial cells throughout human tissues. They function as mechanosensors and contribute to cellular homeostasis through proliferation, differentiation and apoptotic processes. Nevertheless, their fundamental role in cell-to-cell mechanical interactions, cell–matrix communication, tissue morphogenesis and planar cell polarity connote their engagement in processes of oncogenesis, including invasion and metastasis. The aim of the study was to investigate the expression of PC-1 and PC-2 in colorectal cancer (CRC), identify possible correlations with histopathological characteristics and explore the underpinning signal transduction mechanisms. Methods The immunohistochemical expression of PC-1 and PC-2 was evaluated in paraffin sections of 144 CRC patients and the results were statistically correlated with clinical and pathologic parameters. Molecular mechanisms were explored by Western blot, immunofluorescence and flow cytometry in SW480 CRC cell line expressing PC-1 and PC-2. Results Significantly elevated cytoplasmic expression was observed for PC-1 and PC-2 in mucinous carcinomas (p = 0.0035 and p = 0.0001, respectively). High expression levels of PC-2 were correlated with advanced TNM stage (p = 0.0324) and depth of tumour invasion (p = 0.0311). High expression levels of PC-1 (p = 0.0078) and altered expression levels of PC-2 in the transition zone from normal to cancerous tissue (p = 0.0198) were associated with reduced five-year survival rate. Altered expression of PC-1 in the transition zone was correlated with higher chance of relapse for the patients (p = 0.0019). Experiments in SW480 cells revealed funneling of PC-1 and PC-2 function via mammalian target of rapamycin (mTOR) and p70S6 kinases. Conclusions Our findings suggest that PC-1 and PC-2 are implicated in the development of an aggressive phenotype in CRC, as well as in reduced five-year survival rate and cancer-free survival, thus constituting potential prognostic markers. Their function seems to be partially mediated by key molecules of the mTOR signaling pathway. Disclosure All authors have declared no conflicts of interest.

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH-dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.