BackgroundPeptidylarginine deiminase type 4 (PAD4/PADI4) post-translationally converts peptidylarginine to citrulline. Recent studies suggest that PADI4 represses expression of p53-regulated genes via citrullination of histones at gene promoters.MethodsExpression of PADI4 was investigated in various tumors and non-tumor tissues (n = 1673) as well as in A549, SKOV3 and U937 tumor cell lines by immunohistochemistry, real-time PCR, and western blot. Levels of PADI4 and citrullinated antithrombin (cAT) were investigated in the blood of patients with various tumors by ELISA (n = 1121).ResultsImmunohistochemistry detected significant PADI4 expression in various malignancies including breast carcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cancer cells, colorectal adenocarcinomas, renal cancer cells, ovarian adenocarcinomas, endometrial carcinomas, uterine adenocarcinomas, bladder carcinomas, chondromas, as well as other metastatic carcinomas. However, PADI4 expression was not observed in benign leiomyomas of stomach, uterine myomas, endometrial hyperplasias, cervical polyps, teratomas, hydatidiform moles, trophoblastic cell hyperplasias, hyroid adenomas, hemangiomas, lymph hyperplasias, schwannomas, neurofibromas, lipomas, and cavernous hemangiomas of the liver. Additionally, PADI4 expression was not detected in non-tumor tissues including cholecystitis, cervicitis and synovitis of osteoarthritis, except in certain acutely inflamed tissues such as in gastritis and appendicitis. Quantitative PCR and western blot analysis showed higher PADI4 expression in gastric adenocarcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cell cancers and breast cancers (n = 5 for each disease) than in the surrounding healthy tissues. Furthermore, western blot analysis detected PADI4 expression in cultured tumor cell lines. ELISA detected increased PADI4 and cAT levels in the blood of patients with various malignant tumors compared to those in patients with chronic inflammation and benign tumors. This was consistent with immunohistochemical results. Additionally, PADI4 and cAT levels were significantly associated with higher levels of known tumor markers.ConclusionOur results suggest that PADI4 expression is increased in the blood and tissues of many malignant tumors, a finding useful for further understanding of tumorigenesis.

SummarySomatic mutations and large-scale depletion in mitochondrial DNA (mtDNA) have been extensively detected in various human cancers. However, it still remains unclear whether the alterations in mtDNA content are related to the clinicopathological parameters and patient prognosis in breast cancer. In the present study, we analyzed the copy number of mtDNA in 59 cases of invasive breast tumors and paired nontumorous tissues using quantitative real-time PCR. Our data showed that the level of mtDNA was significantly decreased in tumor tissues as compared to the adjacent nontumorous counterparts (P ¼ 0.001). The reduced copy number in mtDNA was associated with an older onset age ( 50 years old, P ¼ 0.035) as well as a higher histological grade (P ¼ 0.012). Survival analysis measured by the Kaplan-Meier curves and the log-rank test indicated that patients with reduced mtDNA content had significantly poorer disease-free survival (DFS, P ¼ 0.0079) and overall survival (OS, P ¼ 0.011) rate. In addition, tumors harboring mutations in displacement (D)-loop region, particularly at the polycytidine stretch (T/N ratio ¼ 64.3 + 8.2%) or close to the replication origins of the heavy-strand (T/N ratio ¼ 68.7 + 5.5%), had a significantly lower copy number of mtDNA than the ones without D-loop alterations. Together, our results suggested that reduced copy number of mtDNA may be involved in breast neoplastic transformation or progression and mtDNA content might be potentially used as a tool to predict prognosis. Somatic mutation in the D-loop region probably is one of key contributing factors leading to decreased mtDNA level in breast tumors.

We examined the effect of glucocorticoid agonists on the extinction of conditioned fear in rats by using fear-potentiated startle. Systemic injection of glucocorticoid receptor agonists dexamethasone (DEX) (0.1, 0.5, and 1.0 mg/kg) and intra-amygdala infusion of RU28362 (0.5, 1.0, and 3.0 ng/side) prior to extinction training facilitated extinction of conditioned fear in a dose-dependent manner. Extinction of conditioned fear and circulating corticosterone levels were attenuated by administration of corticosteroid synthesis inhibitor metyrapone (25 mg/kg s.c.) 90 min before extinction training. The facilitation effect of DEX was dependent on repeated presentation of the conditioned stimulus rather than exposure to the experimental context, indicating this effect did not result from impaired expression of conditioned fear or accelerated forgetting. Intra-amygdaloid administration of the glucocorticoid receptor antagonist mifepristone (0.1, 0.2, and 0.5 ng/side, bilaterally) blocked extinction of conditioned fear and the facilitation effect of DEX in a dose-dependent manner. Mifepristone (2 ng/side) did not affect extinction but blocked the facilitating effect of DEX. Systemic administration of DEX after extinction training also facilitated extinction, suggesting that DEX may influence the memory consodilation phase of extinction. The Dose of dexamethsone or metyrapone used here did not influence fear-potentiated startle when administered before testing. Thus, it is unlikely that these drugs influenced extinction by increasing or disrupting CS processing. All results suggested that amygdaloid glucocorticoid receptors were involved in the extinction of conditioned fear. Neuropsychopharmacology (2006) 31, 912-924.

Alzheimer disease (AD) is an age-dependent neurodegenerative disease characterized by the formation of β–amyloid (Aβ)-containing senile plaque. The disease could be induced by the administration of Aβ peptide, which was also known to upregulate inducible nitric oxide synthase (iNOS) and stimulate neuronal apoptosis. The present study is aimed to elucidate the cellular effect of resveratrol, a natural phytoestrogen with neuroprotective activities, on Aβ-induced hippocampal neuron loss and memory impairment. On adult Sprague-Dawley rats, we found the injection of Aβ could result in a significant impairment in spatial memory, a marked increase in the cellular level of iNOS and lipid peroxidation, and an apparent decrease in the expression of heme oxygenase-1 (HO-1). By combining the treatment with Aβ, resveratrol was able to confer a significant improvement in spatial memory, and protect animals from Aβ-induced neurotoxicity. These neurological protection effects of resveratrol were associated with a reduction in the cellular levels of iNOS and lipid peroxidation and an increase in the production of HO-1. Moreover, the similar neurological and cellular response were also observed when Aβ treatment was combined with the administration of a NOS inhibitor, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME). These findings strongly implicate that iNOS is involved in the Aβ-induced lipid peroxidation and HO-1 downregulation, and resveratrol protects animals from Aβ-induced neurotoxicity by suppressing iNOS production.

Resveratrol is a natural polyphenol and possesses many biological functions such as anti-inflammatory activity and protection against atherosclerosis and myocardial infraction. Parkinson's disease is a common progressive neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is the most useful neurotoxin to induce Parkinsonism. The present study was carried out to elucidate the neuroprotective effect and possible mechanism of resveratrol on MPTP-induced striatal neuron loss. Sixty adult Balb/c mice were divided into four groups: sham operation, MPTP treatment (30 mg/kg, i.p.), MPTP combined with resveratrol administration (20 mg/kg, i.v.), and resveratrol treatment alone. Microdialysis and high-performance liquid chromatography were used to analyze dihydroxybenzoic acid (DHBA) that reflected the hydroxyl radical level. In the present study, we found MPTP chronic administration significantly induced motor coordination impairment in mice. After MPTP administration, the hydroxyl radical levels in substantia nigra were also significantly elevated and animals displayed severe neuronal loss. Resveratrol administration significantly protected mice from MPTP-induced motor coordination impairment, hydroxyl radical overloading, and neuronal loss. Our results demonstrated that resveratrol could elicit neuroprotective effects on MPTP-induced Parkinsonism through free radical scavenging.

Context.-Invasive micropapillary carcinoma (IMPC) is a distinct variant of mammary carcinoma in which tumor cells are arranged in morulelike clusters devoid of fibrovascular cores and situated within empty stromal spaces. Identification of IMPC can be achieved by the assessment of morphologic features in conjunction with the characteristic ''inside-out'' staining pattern of epithelial membrane antigen and sialyl Lewis X highlighted by immunohistochemical analysis. Although recognizing micropapillary architecture is often not challenging, the criteria for distinguishing between mixed and pure IMPC remain imprecise. Some mucin-producing carcinomas can also have micropapillary histology, but there is no consensus on whether these tumors are variants of IMPC or mucinous carcinomas. The molecular genetic studies demonstrate that IMPCs have distinct molecular genetic profiles, supporting the theory that they constitute distinct pathologic entities. However, genomic analyses have not identified any specific genomic aberration that may explain the distinctive morphology and clinical behavior of IMPC.Objective.-To provide an overview on the current concepts in the diagnosis and pathogenesis of IMPC of the breast, incorporating recent molecular genetic advances and prognosis-based reclassification.Data Sources.-PubMed search and the cited references were reviewed.Conclusions.-The recent evolution of prognosis-based reclassification and molecular genetic advances has enhanced our knowledge of the pathogenesis of IMPC of the breast. Additional studies might reveal consistent molecular alterations that underlie the formation of the inside-out growth pattern, and they might elucidate the molecular mechanisms responsible for the unfavorable clinical behavior of IMPC.

Interleukin-1 (IL-1) has many roles in the brain in addition to mediating inflammatory processes in the glia, and has also been implicated in neurodegenerative disease. Traumatic brain injury (TBI) is one of the most prevalent causes of morbidity and mortality in young persons. We conducted a study to assess the effect of IL-1 on the TBI-induced death of hippocampal neurons. After TBI was induced in adult male Sprague-Dawley rats under anesthesia, we evaluated neuronal damage score through microscopic examination and Pulsinelli's grading system. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to measure the levels of IL-1alpha and IL- 1beta in brain tissue at different points after the induction of TBI. Over a 4-day period, the specific sites of release of IL-1alpha and IL-1beta in the brain were elucidated by immunocytochemistry with double- labeling. TBI to the hippocampus was followed by disruption of the blood-brain barrier and severe neuronal loss. Levels of IL-1alpha RNA and protein were significantly elevated at 3 h after TBI, peaked at 12 h, and remained elevated for 168 h. IL-1beta RNA and protein expression were also elevated at 3 h after TBI, but remained so only for 48 h. Our findings indicate that the observed TBI-induced increases in IL-1alpha and IL-1beta occur largely through release of these cytokines from neurons and astrocytes, respectively. Intraventricular administration of antibodies to IL-1alpha and IL-1beta before TBI significantly attenuated the TBI-induced loss of hippocampal neurons. These results show that IL-1alpha and IL-1beta play important roles in the TBI-induced loss of hippocampal neurons.

No abstract