Low dietary iron intake restrains the intestinal inflammatory response and pathology of enteric infection by food-borne bacterial pathogens.

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition.

Activation of apoptosis signal regulating kinase 1 (ASK1) and translocation of death-associated protein, Daxx, in substantia nigra pars compacta in a mouse model of Parkinson's disease: protection by alpha-lipoic acid.

Parkinson's disease (PD), a neurodegenerative disorder, causes severe motor impairment due to loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). MPTP, a neurotoxin that causes dopaminergic cell loss in mice, was used in an animal model to study the pathogenic mechanisms leading to neurodegeneration. We observed the activation of apoptosis signal regulating kinase (ASK1, MAPKKK) and phosphorylation of its downstream targets MKK4 and JNK, 12 h after administration of a single dose of MPTP. Further, Daxx, the death-associated protein, translocated to the cytosol selectively in SNpc neurons seemingly due to MPTP mediated down-regulation of DJ-1, the redox-sensitive protein that binds Daxx in the nucleus. Coadministration of alpha-lipoic acid (ALA), a thiol antioxidant, abolished the activation of ASK1 and phosphorylation of downstream kinases, MKK4, and JNK and prevented the down-regulation of DJ-1 and translocation of Daxx to the cytosol seen after MPTP. ALA also attenuated dopaminergic cell loss in SNpc seen after subchronic MPTP treatment. Our studies demonstrate for the first time that MPTP triggers death signaling pathway by activating ASK1 and translocating Daxx, in vivo, in dopaminergic neurons in SNpc of mice and thiol antioxidants, such as ALA terminate this cascade and afford neuroprotection.

[Effect of Coptis Chinensis compound on the gene expression in transplanted tumor tissue in nasopharyngeal carcinoma cell line of CNE1 by cDNA microarray].

OBJECTIVE: To investigate the effect of Coptis Chinensis compound on the gene expression of transplanted tumor in nasopharyngeal carcinoma cell line of CNE1. METHODS: The cells were injected into Balb/C nude mice, and after the transplanted tumor was established, the nude mice with tumors were treated with Coptis chinensis compound. The nude mice were then killed, tumor tissues were isolated and the total RNA was extracted. Hybridization with a cDNA microarray was conducted using a probe of fluorescent-labeled cDNA mixture that was reversely transcribed from the RNA. The gene expression level was analyzed after scanning the fluorescent intensity by a laser scanner, and further confirmed by RT-PCR. RESULTS: After using Coptis Chinensis compound for 30 days, the size of transplanted tumors were remarkably reduced, and the inhibition rate of tumor growth was 29.5%. In the meantime, the expression of 147 genes in the tissue of implanted tumor treated with the compound altered: the expression of 102 genes was down-regulated, and that of 45 genes was up-regulated. Furthermore, the differential expression of 3 genes was verified by RT-PCR, MAD3 and H731 genes were up-regulated, and that of CHK1 was down-regulated. CONCLUSION: Coptis Chinensis compound can affect the gene expression in the tissue of implanted tumor, which implies that the inhibition of Coptis Chinensis compound on the growth of the implanted tumor with CNE1 cells in nude mice might be associated with the control of gene expression.

Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation.

The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1- and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1- and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5' regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.

Cloning and functional expression of rat ether-à-go-go-like K+ channel genes.

1. Screening of rat cortex cDNA resulted in cloning of two complete and one partial orthologue of the Drosophila ether-à-go-go-like K+ channel (elk). 2. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed predominant expression of rat elk mRNAs in brain. Each rat elk mRNA showed a distinct, but overlapping expression pattern in different rat brain areas. 3. Transient transfection of Chinese hamster ovary (CHO) cells with rat elk1 or rat elk2 cDNA gave rise to voltage-activated K+ channels with novel properties. 4. RELK1 channels mediated slowly activating sustained potassium currents. The threshold for activation was at -90 mV. Currents were insensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP), but were blocked by micromolar concentrations of Ba2+. RELK1 activation kinetics were not dependent on prepulse potential like REAG-mediated currents. 5. RELK2 channels produced currents with a fast inactivation component and HERG-like tail currents. RELK2 currents were not sensitive to the HERG channel blocker E4031.

Expression of tyrosine kinase gene in mouse thymic stromal cells.

Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both compartments is required to dissect the mechanisms that control this process. Here we report a search for PTK in mouse TSC, using RT-PCR to survey the repertoire of PTK mRNAs expressed in a freshly isolated TSC preparation. We identified 10 different PTK cDNAs among the 216 cDNAs sequenced, and demonstrate that transcripts of three of those (ufo, fyn and fer) are widely expressed among a large panel of immortalized thymic epithelial cell lines (TEC) and in primary cultures of TSC. Of the other seven, none were expressed in established TEC lines but, instead, displayed distinct expression patterns in cell types likely to have contaminated the fresh TSC preparation, i.e., macrophages, B cells, T cells and fibroblasts. Among the three PTK expressed in TEC lines, only one, ufo, exhibited expression exclusively in cells of non-hemopoietic origin. Although expression of ufo (also known as tyro 7, axl or ark) is not thymic-specific, in that it is also expressed in cell types of mesodermal origin in other tissues, its presence in TEC suggests a role for ufo in differentiation of the TSC compartment. Consistent with this notion, high-level expression of this receptor PTK at the protein level could be documented in every TEC line investigated, as well as in fresh thymus tissue sections. These data provide the first example of a receptor PTK in TSC and open new approaches to study the regulation of TSC differentiation.

Oncoprotein expression in human synovial tissue: an immunohistochemical study of different types of arthritis.

Based on the fact that synovial lining cells have some properties of transformed-appearing cells, we have examined the expression of Myc, Myb, Fos, Jun and Ras oncoproteins in synovial tissues from patients with different types of arthritis. Formalin-fixed and paraffin-embedded sections of synovial tissue from 12 patients with rheumatoid arthritis (RA), 14 with reactive arthritis (ReA), nine with other seronegative arthritis (OSA), seven with bacterial arthritis (BA), eight with probable bacterial arthritis (PBA) and eight with osteoarthritis (OA) were studied using the immunoperoxidase staining technique. The oncoproteins studied were expressed both in the synovial lining layer and in the sublining layer, consisting of lymphocytes, other inflammatory cells and blood vessels. Among the six disease entities, RA and OA appeared to be the most distinct, whereas the results obtained for ReA and OSA, and on the other hand for BA and PBA, closely resembled each other. The expression of Myc, Myb, Fos and Jun was significantly correlated both to the degree of synovial hypercellularity and the synovial lymphocytic infiltration. For Ras, such a correlation could not be seen. We conclude that we find no evidence of a cell lineage-specific or a disease-specific abnormality of proto-oncogene products in RA, and the expression of these oncoproteins is consistent with inflammation rather than with any primary abnormality of cell growth.

Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line.

BACKGROUND: Alterations in the expression of genes that control the cell cycle may be of critical importance in determining the sensitivity of cells and tumors to drugs (chemosensitivity) and radiation. Mutations and deletions of the p53 tumor suppressor gene in cell lines and tumors are associated with resistance to a variety of DNA-damaging agents. The effects of alterations in the cyclin genes and their products on drug action have not been studied. One of these genes, cyclin D1, is expressed in early G1 phase, and its protein product, together with the cyclin-dependent kinases CDK4 and CDK6, mediates the phosphorylation and functional inactivation of the retinoblastoma protein (pRb). Elevated levels of expression of cyclin D1 protein have been found in a variety of cancers, including breast cancer, head and neck cancer, non-small-cell lung cancer, and mantle cell lymphomas. PURPOSE: This study was conducted to investigate the effect of increased expression of cyclin D1 protein on the chemosensitivity profile of a human fibrosarcoma cell line. METHODS: Expression plasmids containing either the neomycin-resistance gene and the complementary DNA sequence encoding human cyclin D1 or the neomycin-resistance gene only (control) were transfected by lipofection into the human HT1080 fibrosarcoma cell line, and cell colonies resistant to the antibiotic neomycin (G418) were isolated. Cyclin D1 messenger RNA (mRNA) and protein levels were measured by ribonuclease protection and western blot analyses, respectively. Dihydrofolate reductase (DHFR) mRNA and protein levels were measured by northern blot and western blot analyses, respectively. The phosphorylation status of pRb was assessed by western blot analysis. Cell cycle analysis was performed by use of the technique of fluorescence-activated cell sorting. Cytotoxicity assays were carried out by use of the sulforhodamine blue assay. RESULTS: Of the 16 cyclin D1-transfected cell clones that were isolated, four were randomly selected for further study. Two cell clones expressed high levels of cyclin D1 mRNA and protein as compared with control cells transfected with plasmids containing the neomycin-resistance gene only. A relative increase in the phosphorylated form of pRb in cells expressing high versus low levels of cyclin D1 was also revealed by western blot analysis. There was an increased fraction of cells in the S and G2 phases of the cell cycle among cells expressing higher levels of cyclin D1. Transfectants with increased cyclin D1 expression also had increased DHFR mRNA and protein expression. Cytotoxicity assays revealed a statistically significant (P < .01) increase in resistance to methotrexate in cells expressing high levels of cyclin D1 compared with cells expressing lower levels. There was no difference in resistance to doxorubicin, paclitaxel (Taxol), and cytarabine. CONCLUSION: Alterations in the expression of cyclin D1 led to altered cell cycle distribution in a human sarcoma cell line. The associated increase in DHFR expression resulted in increased resistance to methotrexate but had no effect on other classes of anticancer agents. IMPLICATIONS: These results indicate that alterations in cell cycle genes may differ in their effects on cytotoxicity. It will be important to determine the effects of alterations of other cell cycle regulatory genes on the responses of cells to specific classes of drugs. Tumors with overexpression of cyclin D1 may be relatively refractory to methotrexate treatment.

Growth factor regulation of cyclin D1 mRNA expression through protein synthesis-dependent and -independent mechanisms.

Overexpression of the cyclin D1/PRAD1 oncogene has been observed in a number of tumorigenic cell lines, suggesting that regulation of D1 expression may represent an important step in the control of cellular proliferation. We have examined the mRNA expression of cyclin D1, as well as two related D-type cyclins, D2 and D3, in response to defined growth factors that control the growth of Balb/c-3T3 fibroblasts. Transcripts for all three D-type cyclins were expressed during the G1 phase of the Balb cell cycle, however only D1 and D3 exhibited periodic induction. Although redundantly expressed, message levels of cyclin D1 and D3 were differentially regulated in regard to kinetics of induction; a modest increase in D3 mRNA was detected near the G1/S boundary, 12 h after serum stimulation of quiescent cells, while abundance of D1 transcript increased 20 to 30-fold, peaking 6 h after addition of serum. Factors such as platelet-derived growth factor (PDGF) that induce competence formation in Balb cells, increased D1 message and protein levels to the same extent as serum but did not affect expression of cyclin D3 and did not stimulate entry into S phase. Progression factors contained within platelet-poor plasma stimulated D1 expression only weakly but acted synergistically with low concentrations of PDGF to increase D1 mRNA to maximum levels. Depletion of protein kinase C severely reduced the ability of PDGF and serum to induce D1 mRNA. PDGF- and serum-mediated elevation of steady-state D1 message levels was in part because of a transcriptional activation of the D1 gene that was independent of protein synthesis. However, protein synthesis was required 3-4 h after serum stimulation for the shut down of D1 transcription leading to the normal decline in message levels after peak induction. Our results indicate that overexpression of cyclin D1 message may result from a disruption of negative regulatory events that repress D1 transcription.

Differential expression of transporters for norepinephrine and glutamate in wild type, variant, and WNT1-expressing PC12 cells.

Wild type PC12 pheochromocytoma cells express a Na(+)-dependent norepinephrine transporter that operates in the uptake of catecholamines, including dopamine. This transporter is not expressed in two spontaneously occurring flat cell variants of PC12 or in two other flat cell variants whose phenotype was induced by expression of the Wnt-1 oncogene. However, each of the flat cell variants, including those that express Wnt-1, exhibit a Na(+)-dependent, Cl(-)-independent glutamate/aspartate transporter activity that is not present in wild type PC12 cells. The flat cell variants took up glycine by a Na(+)-dependent process as well as did wild type cells. All of the flat cell variants have decreased levels of norepinephrine transporter mRNA but normal levels of glycine transporter mRNA. Glutamate/aspartate transporter mRNA was detected only in the variants that exhibited glutamate/aspartate transporter activity, and the nucleotide sequence of a partial glutamate/aspartate transporter cDNA from these cells demonstrated that it was the glial form of the transporter that was expressed. These variants were more sensitive than was wild type PC12 to alanosine, a toxic aspartate analog that enters cells by a transporter-mediated system such as the glutamate/aspartate transporter; however, these variants were as sensitive as wild type cells to another toxic aspartate analog, N-(phosphonacetyl)-L-aspartic acid, which is believed to enter cells by endocytosis. We suggest that the Wnt-1 gene product, or a homolog, may be involved in glial differentiation and that the mechanisms that alter the expression of the norepinephrine and glutamate/aspartate transporters in wild type and variant PC12 cells may also operate to regulate neurotransmitter transporter expression in vivo.