Interconversion of estrone and estradiol-17β in lung slices of the adult human

The metabolism of tritium-labeled estrone and estradiol-17β in slices of lung tissue obtained from an adult human was studied: estrone was identified as the only metabolite of estradiol-17β and estradiol-17β as the exclusive product of estrone metabolism. Product formation remained linear as a function of time of incubation up to 3 h and of wet lung tissue mass up to 300 mg/ml. At equimolar substrate concentrations, the rates of estrone formation were at least 2-fold greater than those of estradiol-17β. The apparent K_M of 17β-hydroxysteroid oxidoreductase for estrone was 11 μM and that for estradiol-17β was 10 μM. These results are suggestive that the human lung enzyme binds estrone and estradiol-17β with similar affinities; however, the oxidative pathway is favored as indicated by the greater V_max attained in the formation of estrone. It is possible that, in vivo, the human lung constitutes a site for estradiol-17β inactivation to estrone as well as a site for the conversion of estrone to estradiol-17β. This last process may become particularly important in instances in which the ovaries have ceased to function and secrete estradiol-17β, e.g. the postmenopausal women.     

Amyloid-β oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons

Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer''s disease (AD). Soluble amyloid-β oligomers (AβOs), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. AβOs also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that AβOs reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3β, downstream targets of CaN, prevents BDNF transport defects induced by AβOs. We further show that AβOs induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for AβO-induced BDNF transport disruption.     

Amyloid-β annular protofibrils evade fibrillar fate in Alzheimer disease brain

Annular protofibrils (APFs) represent a new and distinct class of amyloid structures formed by disease-associated proteins. In vitro, these pore-like structures have been implicated in membrane permeabilization and ion homeostasis via pore formation. Still, evidence for their formation and relevance in vivo is lacking. Herein, we report that APFs are in a distinct pathway from fibril formation in vitro and in vivo. In human Alzheimer disease brain samples, amyloid-β APFs were associated with diffuse plaques, but not compact plaques; moreover, we show the formation of intracellular APFs. Our results together with previous studies suggest that the prevention of amyloid-β annular protofibril formation could be a relevant target for the prevention of amyloid-β toxicity in Alzheimer disease.     

Amyloid-β forms fibrils by nucleated conformational conversion of oligomers

Amyloid-β amyloidogenesis is reported to occur via a nucleated polymerization mechanism. If this is true, the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many laboratories have detected early nonfibrillar amyloid-β oligomers without observing amyloid fibrils, suggesting that a mechanistic revision may be needed. Here we introduce Cys-Cys-amyloid-β(1-40), which cannot bind to the latent fluorophore FlAsH as a monomer, but can bind FlAsH as an nonfibrillar oligomer or as a fibril, rendering the conjugates fluorescent. Through FlAsH monitoring of Cys-Cys-amyloid-β(1-40) aggregation, we found that amyloid-β(1-40) rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism. This methodology was used to show that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-associated oligomerization phase of amyloid-β amyloidogenesis while allowing fibril formation, rationalizing how low concentrations of plasmalogen ethanolamine in the brain are epidemiologically linked to Alzheimer's disease.     

Tissue differential expression of lycopene β-cyclase gene in papaya

Carotene pigments in flowers and fruits are distinct features related to fitness advantages such as attracting insects for pollination and birds for seed dispersal. In papaya, the flesh color of the fruit is considered a quality trait that correlates with nutritional value and is linked to shelf-life of the fruit. To elucidate the carotenoid biosynthesis pathway in papaya, we took a candidate gene approach to clone the lycopene β-cyclase gene, LCY-B. A papaya LCY-B ortholog, cpLCY-B, was successfully identified from both cDNA and bacterial artificial chromosome （BAC） libraries and complete genomic sequence was obtained from the positive BAC including the promoter region. This cpLCY-B shared 80% amino acid identity with citrus LCY-B. However, full genomic sequences from both yellow- and red-fleshed papaya were identical. Quantitative real-time PCR （qPCR） revealed similar levels of expression at six different maturing stages of fruits for both yellow- and red-fleshed genotypes. Further expression analyses of cpLCY-B showed that its expression levels were seven- and three-fold higher in leaves and, respectively, flowers than in fruits, suggesting that cpLCY-B is down-regulated during the fruit ripening process.     

Does neonatal brain ischemia induce schizophrenia-like behavior in young adult rats?

Perinatal cerebral hypoxia represents a major cause of obstetric complications and the resulting transient oxygen deficiency might belong to early risk factors for schizophrenia. The aim of this study was to evaluate possible long-term behavioral changes induced by one hour of continuous bilateral common carotid artery occlusion in 12-day-old male rats. Post-ischemic behavioral disturbances were evaluated in social (play) behavior on postnatal day 22 (PND 22), open field test (PND 35 and 50) and prepulse inhibition of the acoustic startle reflex (PND 50). Transient ischemia in neonatal rats was not significantly altered in social dyadic interactions evaluated in pre-weaning pups, but resulted in enhanced locomotor activity in pubertal rats (PND 35) and impaired prepulse inhibition of the startle reflex in post-pubertal males (PND 50). These behavioral alterations suggest that perinatal hypoxic/ischemic insults may represent a risk factor for later manifestation of specific features relevant to schizophrenia in predisposed individuals.     

Involvement of TNF-α in differential gene expression pattern of CXCR4 on human marrow-derived mesenchymal stem cells

Cell therapy and tissue repair are used in a variety of diseases including tissue and organ transplantation, autoimmune diseases and cancers. Now mesenchymal stem cells (MSCs) are an attractive and promising source for cell-based therapy according to their individual characteristics. Soluble factors which are able to induce MSCs migration have a vital role in cell engraftment and tissue regeneration. Tumor necrosis factor α (TNF-α) is a major cytokine present in damaged tissues. We have investigated the pattern of gene expression of chemokine receptor CXCR4 in nine groups of human bone marrow-derived MSCs stimulated with TNF-α in different dose and time manner. Comparison of TNF-α treated with untreated MSCs revealed the highest expression level of CXCR4 after treatment with 1, and 10 ng/ml of TNF-α in 24 h, and the production of CXCR4 mRNA was regulated up to 216 and 512 fold, respectively. Our results demonstrated the differential gene expression pattern of chemokine receptor CXCR4 in human marrow-derived MSCs stimulated with inflammatory cytokine TNF-α. These findings suggest that in vitro control of both dose and time factors may be important in stem cell migration capacity, and perhaps in future-stem cell transplantation therapies.     

Differential regulation of IFNα, IFNβ and IFNε gene expression in human cervical epithelial cells

Interferonε (IFNε) is a unique type I IFN that has distinct functions from IFNα/β. IFNε is constitutively expressed at mucosal tissues, including the female genital mucosa, and is reported to be modulated by estrogen and seminal plasma. However, its regulation by cytokines, including TNFα, IL-1β, IL-6, IL-8, IL-17, IL-22 and IFNα, which are commonly present in the female genital mucosa, is not well documented in freshly isolated primary cervical cells from tissues. We determined the effect of these cytokines on gene expression of type I IFNs in an immortalized endocervical epithelial cell line (A2EN) and in primary cervical epithelial cells. Several pro-inflammatory cytokines were found to induce IFNε, and TNFα induced the strongest response in both cell types. Pretreatment of cells with the IκB inhibitor, which blocks the NF-κB pathway, suppressed TNFα-mediated IFNε gene induction and promoter activation. Expression of IFNα, IFNβ, and IFNε was differentially regulated in response to various cytokines. Taken together, our results show that regulation of these IFNs depends on cell type, cytokine concentration, and incubation time, highlighting the complexity of the cytokine network in the cervical epithelium.     

Tumor-produced, active interleukin-1β regulates gene expression in carcinoma-associated fibroblasts

Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial–mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1β (IL1-β) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-β expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-β processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-β. IL1-β signaling was investigated by western blot and immunocytochemistry. IL1-β-regulated genes were analyzed by real-time qPCR.SCC-25 cells produced 16 kD active IL1-β, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NFκBα. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-β reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-β-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-β in the tumor cells leads to IL1-β-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression.     

TGF-β polymorphism and its expression correlated with CXCR4 expression in human breast cancer

The role of chemokines and the growth factors has been extensively analyzed both in cancer risk and tumor progression. The transforming growth factor beta (TGF-β) and chemokine (C-X-C motif) receptor 4 (CXCR4) genes are implicated in several diseases, including breast cancer. Genomic DNA was obtained from 21 samples of peripheral blood or from normal tissue, previously fixed in formalin and embedded in paraffin for TGF-β T869C polymorphism analyses. Total cellular RNA was extracted from the same 21 patients, but from fresh tissue (tumor and adjacent healthy from the same breast) for expression analysis by Real Time PCR. No significant differences were observed in genotype distribution according to clinicopathological characteristics. Transforming growth factor beta (TGF-β) mRNA expression was assessed according to T869C polymorphism and CC patients presented a higher TGF-β expression but not significant when compared to other genotypes (p?=?0.064). A positive correlation was observed in relative mRNA expressions of CXCR4 and TGF-β (p?=?0.020). It is known that overexpression of TGF-β by both tumor and stromal tissue can facilitate the development of metastases, mainly by TGF-β stimulated angiogenesis and increased tumor cell motility. Our findings suggested a role of these genes as progression markers for breast carcinoma.