Aspartyl Asparaginyl Β-hydroxylase Research Articles

Ethanol consumption during gestation promotes placental alterations in IGF-1 deficient placentas

Background During pregnancy, the placenta is an extremely important organ as it secretes its own hormones, e.g. insulin-like growth factor 1 (IGF-1), to ensure proper intrauterine fetal growth and development. Ethanol, an addictive and widely used drug, has numerous adverse effects during pregnancy, including fetal growth restriction (FGR). To date, the molecular mechanisms by which ethanol triggers its toxic effects during pregnancy, particularly in the placenta, are not entirely known. For this reason, a murine model of partial IGF-1 deficiency was used to determine ethanol alterations in placental morphology and AAH expression. Methods Heterozygous (HZ, Igf1+/- ) female mice were given 10% ethanol during 14 days as an acclimation period and throughout pregnancy. HZ female mice given water were used as controls. At gestational day 19, pregnant dams were sacrificed, placentas were collected and genotyped for subsequent studies. Results IGF-1 deficiency and ethanol consumption during pregnancy altered placental morphology, and decreased placental efficiency and aspartyl/asparaginyl β-hydroxylase (AAH) expression in placentas from all genotypes. No differences were found in Igf1, Igf2, Igf1r and Igf2r mRNA expression in placentas from all groups. Conclusions IGF-1 deficiency and ethanol consumption throughout gestation altered placental development, suggesting the crucial role of IGF-1 in the establishment of an adequate intrauterine environment that allows fetal growth. However, more studies are needed to study the precise mechanism to stablish the relation between both insults.

Modification of Regulatory T Cell Epitopes Promotes Effector T Cell Responses to Aspartyl/Asparaginyl β-Hydroxylase.

Cancer is a leading cause of death worldwide. The search for innovative therapeutic approaches is a principal focus of medical research. Vaccine strategies targeting a number of tumor-associated antigens are currently being evaluated. To date, none have garnered significant success. Purportedly, an immunosuppressive tumor microenvironment and the accumulation of regulatory T cells contribute to a lack of tumor vaccine efficacy. Aspartyl/asparaginyl β-hydroxylase (ASPH), a promising therapeutic target, is overexpressed in a variety of malignant tumors but is expressed negligibly in normal tissues. Computer analysis predicted that ASPH expresses four peptide sequences (epitopes) capable of stimulating regulatory T cell activity. The abolition of these putative regulatory T cell epitopes increased the CD4+ and CD8+ effector T cell responses to monocyte-derived dendritic cells pulsed with a modified, epitope-depleted version of ASPH in an ex vivo human lymphoid tissue-equivalent coculture system while simultaneously decreasing the overall number of FoxP3+ regulatory T cells. These findings suggest that the efficacy of all new vaccine candidates would profit from screening and eliminating potential tolerogenic regulatory T cell epitopes.

Dietary Soy Prevents Alcohol-Mediated Neurocognitive Dysfunction and Associated Impairments in Brain Insulin Pathway Signaling in an Adolescent Rat Model.

Background: Alcohol-related brain degeneration is linked to cognitive-motor deficits and impaired signaling through insulin/insulin-like growth factor type 1 (IGF-1)-Akt pathways that regulate cell survival, plasticity, metabolism, and homeostasis. In addition, ethanol inhibits Aspartyl-asparaginyl-β-hydroxylase (ASPH), a downstream target of insulin/IGF-1-Akt signaling and an activator of Notch networks. Previous studies have suggested that early treatment with insulin sensitizers or dietary soy could reduce or prevent the long-term adverse effects of chronic ethanol feeding. Objective: The goal of this study was to assess the effects of substituting soy isolate for casein to prevent or reduce ethanol’s adverse effects on brain structure and function. Methods: Young adolescent male and female Long Evans were used in a 4-way model as follows: Control + Casein; Ethanol + Casein; Control + Soy; Ethanol + Soy; Control = 0% ethanol; Ethanol = 26% ethanol (caloric). Rats were fed isocaloric diets from 4 to 11 weeks of age. During the final experimental week, the Morris Water maze test was used to assess spatial learning (4 consecutive days), after which the brains were harvested to measure the temporal lobe expression of the total phospho-Akt pathway and downstream target proteins using multiplex bead-based enzyme-linked immunosorbent assays (ELISAs) and duplex ELISAs. Results: Ethanol inhibited spatial learning and reduced brain weight, insulin signaling through Akt, and the expression of ASPH when standard casein was provided as the protein source. The substitution of soy isolate for casein largely abrogated the adverse effects of chronic ethanol feeding. In contrast, Notch signaling protein expression was minimally altered by ethanol or soy isolate. Conclusions: These novel findings suggest that the insulin sensitizer properties of soy isolate may prevent some of the adverse effects that chronic ethanol exposure has on neurobehavioral function and insulin-regulated metabolic pathways in adolescent brains.

Ethanol consumption during gestation promotes placental alterations in IGF-1 deficient mice

Background: During pregnancy, the placenta is an extremely important organ as it secretes its own hormones, e.g. insulin-like growth factor 1 (IGF-1), to ensure proper intrauterine fetal growth and development. Ethanol, an addictive and widely used drug, has numerous adverse effects during pregnancy, including fetal growth restriction (FGR). To date, the molecular mechanisms by which ethanol triggers its toxic effects during pregnancy, particularly in the placenta, are not entirely known. For this reason, a murine model of partial IGF-1 deficiency was used to determine ethanol alterations in placental morphology and AAH expression. Methods: Heterozygous (HZ, Igf1+/-) female mice were given 10% ethanol during 14 days as an acclimation period and throughout pregnancy. HZ female mice given water were used as controls. At gestational day 19, pregnant dams were sacrificed, placentas were collected and genotyped for subsequent studies. Results: IGF-1 deficiency and ethanol consumption during pregnancy altered placental morphology, and decreased placental efficiency and aspartyl/asparaginyl β-hydroxylase (AAH) expression in placentas from all genotypes. No differences were found in Igf1, Igf2, Igf1r and Igf2r mRNA expression in placentas from all groups. Conclusions: IGF-1 deficiency and ethanol consumption throughout gestation altered placental development, suggesting the crucial role of IGF-1 in the establishment of an adequate intrauterine environment that allows fetal growth. However, more studies are needed to study the precise mechanism to stablish the relation between both insults.

Early diagnosis of prostate cancer, AABH (Aspartyl Asparaginyl β-hydroxylase), a predictive biomarker: A serum immunoassay and personalized medicine.

353 Background: Prostate Cancer (PC) is the most common form of cancer in men in the United States. In their lifetime, approximately 1 out of every 6 American men will develop PC. The chances of developing PC increase dramatically after age 50 with more than 70% of cases occurring in individuals over 65 years of age. Methods: We have investigated the use of aspartyl (asparaginyl) β-hydroxylase (AABH) as a cancer biomarker. We have developed a sandwich ELISA for its detection. Here this assay was utilized to screen sera from patients with prostate cancer and PSA values in various ranges. Results: AABH levels were assessed in sera from 263 individuals with PC and 73 men over 50 years of age not known to have cancer. The levels of AABH in the two groups were >3.00 ng/ml and <3.00 ng/ml, respectively. The overall sensitivity and specificity of the test were 97.5% and 96%. AABH and PSA levels were also compared. The AABH values and sensitivities in individuals with PC and PSA values in the ranges <2 ng/ml, 2-4 ng/ml and >4 ng/ml were >3.00 ng/ml, 99% (n=115); > 3.00 ng/ml, 98% (n=64) and >3.00 ng/ml, 95% (n=114). Conclusions: The present study of AABH indicates that cancer group can be clearly distinguished from normal healthy controls. Moreover, the study indicates that AABH might be used to identify disease in individuals with questionable PSA values. These results support to the establishment of routine screening of serum AABH as a means to detect those who need referral for prostate biopsy. The use of AABH testing may enhance current prostate cancer screening practices by both identifying individuals with low but suspicious PSA values who should be recommended for biopsy and reducing the number of false positive biopsies indicated by positive PSA tests. Serum AABH levels are significantly elevated in individuals with prostate cancer. Using a cutoff value of 3ng/ml, overall sensitivity of the assay for prostate cancer is 97%. The test has a specificity of 96% in healthy men over 50 years of age. AABH is elevated in prostate cancer tissue but NOT in normal prostate or in Benign Prostatic Hyperplasia (BPH). Serum AABH levels are NOT correlate with PSA levels.

An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase.

Background and purpose:Aspartyl/asparaginyl β-hydroxylase (ASPH) is abundantly expressed in malignant neoplastic cells. The establishment of a human cell line overexpressing ASPH could provide the native-like recombinant protein needed for developing theranostic probes. In the process of transfection, the obtained cells normally contain a range of cells expressing the different levels of the target of interest. In this paper, we report on our simple innovative approach in the selection of best-transfected cells with the highest expression of ASPH using subclone selection, quantitative real-time polymerase chain reaction, and gradual increment of hygromycin concentration.Experimental approach:To achieve this goal, human embryonic kidney (HEK 293T) cells were transfected with an ASPH-bearing pcDNA3.1/Hygro(+) vector. During antibiotic selection, single accumulations of the resistant cells were separately cultured and the ASPH mRNA levels of each flask were evaluated. The best subclones were treated with a gradually increasing amount of hygromycin. The ASPH protein expression of the obtained cells was finally evaluated using flow cytometry and immunocytochemistry.Findings / Results:The results showed that different selected subclones expressed different levels of ASPH. Furthermore, the gradual increment of hygromycin (up to 400mg/mL) improved the expression of ASPH. The best relative fold change in mRNA levels was 57.59 ± 4.11. Approximately 90.2% of HEKASPH cells overexpressed ASPH on their surface.Conclusion and implications:The experiments indicated that we have successfully constructed and evaluated a recombinant human cell line overexpressing ASPH on the surface. Moreover, our innovative selection approach provided an effective procedure for enriching highly expressing recombinant cells.

Ethanol-Induced White Matter Atrophy Is Associated with Impaired Expression of Aspartyl-Asparaginyl-β-Hydroxylase (ASPH) and Notch Signaling in an Experimental Rat Model.

Alcohol-induced white matter (WM) degeneration is linked to cognitive-motor deficits and impairs insulin/insulin-like growth factor (IGF) and Notch networks regulating oligodendrocyte function. Ethanol downregulates Aspartyl-Asparaginyl-β-Hydroxylase (ASPH) which drives Notch. These experiments determined if alcohol-related WM degeneration was linked to inhibition of ASPH and Notch. Adult Long Evans rats were fed for 3, 6 or 8 weeks with liquid diets containing 26% ethanol (caloric) and in the last two weeks prior to each endpoint they were binged with 2 g/kg ethanol, 3×/week. Controls were studied in parallel. Histological sections of the frontal lobe and cerebellar vermis were used for image analysis. Frontal WM proteins were used for Western blotting and duplex ELISAs. The ethanol exposures caused progressive reductions in frontal and cerebellar WM. Ethanol-mediated frontal WM atrophy was associated with reduced expression of ASPH, Jagged 1, HES-1, and HIF-1α. These findings link ethanol-induced WM atrophy to inhibition of ASPH expression and signaling through Notch networks, including HIF-1α.

Dose effect of gestational ethanol exposure on placentation and fetal growth

IntroductionPrenatal ethanol exposure compromises fetal growth by impairing placentation. Invasive trophoblastic cells, which mediate placentation, express the insulin-IGF regulated gene, aspartyl-asparaginyl β-hydroxylase (ASPH), which has a critical role in cell motility and invasion. The aims of this study were to characterize effects of ethanol on trophoblastic cell motility, and assess ethanol dose-dependent impairments in placentation and fetal development. MethodsPregnant Long Evans dams were fed with isocaloric liquid diets containing 0%, 8%, 18% or 37% ethanol (caloric content) from gestation day (GD) 6 to GD18. Fetal development, placental morphology, density of invasive trophoblasts at the mesometrial triangle, as well as placental and mesometrial ASPH and Notch-1 protein expression were evaluated. Directional motility of control and ethanol-exposed HTR-8/SVneo cells was assessed by ATP Luminescence-Based assay. ResultsSeverity of fetal growth impairment correlated with increasing doses of ethanol. Ethanol exposure produced dose-dependent alterations in branching morphogenesis at the labyrinthine zone, and inhibited physiological transformation of maternal arteries. ASPH and Notch-1 protein expression levels were reduced, corresponding with impairments in placentation. DiscussionPrenatal ethanol exposure compromises fetal growth and placentation in a dose-responsive manner. Ethanol's adverse effects on placental development are mediated by: 1) altered branching morphogenesis in labyrinthine zone; 2) suppression of invasive trophoblastic precursor cells; and 3) inhibition of trophoblastic cell adhesion and motility, corresponding with reduced ASPH and Notch-1 protein expression.

Mutations in ASPH Cause Facial Dysmorphism, Lens Dislocation, Anterior-Segment Abnormalities, and Spontaneous Filtering Blebs, or Traboulsi Syndrome

We have previously described a syndrome characterized by facial dysmorphism, lens dislocation, anterior-segment abnormalities, and spontaneous filtering blebs (FDLAB, or Traboulsi syndrome). In view of the consanguineous nature of the affected families and the likely autosomal-recessive inheritance pattern of this syndrome, we undertook autozygosity mapping and whole-exome sequencing to identify ASPH as the disease locus, in which we identified two homozygous mutations. ASPH encodes aspartyl/asparaginyl β-hydroxylase (ASPH), which has been found to hydroxylate aspartic acid and asparagine residues on epidermal growth factor (EGF)-domain-containing proteins. The truncating and missense mutations we identified are predicted to severely impair the enzymatic function of ASPH, which suggests a possible link to other forms of ectopia lentis given that many of the genes implicated in this phenotype encode proteins that harbor EGF domains. Developmental analysis of Asph revealed an expression pattern consistent with the proposed link to the human syndrome. Indeed, Asph-knockout mice had a foreshortened snout, which corresponds to the facial abnormalities in individuals with Traboulsi syndrome. These data support a genetic basis for a syndromic form of ectopia lentis and the role of aspartyl hydroxylation in human development.

PPAR Agonist Effects on Notch Signaling Mediators in Experimental Chronic Alcohol-Induced Steatohepatitis

Background: Aspartyl-Asparaginyl-Β-Hydroxylase (ASPH) is a downstream target of insulin and IGF signaling and promotes cell motility for liver remodeling and repair. ASPH functions in part by activating Notch and HIF-1α. PPAR agonists can ameliorate steatohepatitis, hepatic insulin resistance, and reduced ASPH expression in experimental alcoholic liver disease. Herein, we examine the effects of PPAR-α, PPAR-δ, and PPAR-γ agonists on Notch and HIF-1α signaling. Methods: Long Evans rats were chronically fed control or ethanol-containing diets and treated with vehicle, or a PPAR-α, PPAR-δ, or PPAR-γ agonist. ASPH, Notch, and HIF-1α-related genes and proteins were measured in liver. Results: ASPH, Notch, and HIF-1α signaling genes and/or proteins were inhibited by chronic ethanol feeding. PPAR-δ and/or PPAR-γ agonists normalized ASPH, HIF-1α, and PCNA protein in ethanol-exposed livers. In contrast, Notch signaling through HES-1 was not restored. Conclusion: Therapeutic effects of PPAR-δ and PPAR-γ agonists in alcoholic liver disease are mediated by post-translational mechanisms that bolster ASPH-HIF-1α signaling. Alternative strategies are needed to circumvent ethanol-mediated uncoupling of cross-talk among insulin/IGF-1/ASPH-Notch networks.

Metabolic Clues: Novel Directives for Broad Treatment Strategies

Metabolic Clues: Novel Directives for Broad Treatment Strategies