Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

Learning reward timing in cortex through reward dependent expression of synaptic plasticity.

The ability to represent time is an essential component of cognition but its neural basis is unknown. Although extensively studied both behaviorally and electrophysiologically, a general theoretical framework describing the elementary neural mechanisms used by the brain to learn temporal representations is lacking. It is commonly believed that the underlying cellular mechanisms reside in high order cortical regions but recent studies show sustained neural activity in primary sensory cortices that can represent the timing of expected reward. Here, we show that local cortical networks can learn temporal representations through a simple framework predicated on reward dependent expression of synaptic plasticity. We assert that temporal representations are stored in the lateral synaptic connections between neurons and demonstrate that reward-modulated plasticity is sufficient to learn these representations. We implement our model numerically to explain reward-time learning in the primary visual cortex (V1), demonstrate experimental support, and suggest additional experimentally verifiable predictions.

Activation of heat shock and antioxidant responses by the natural product celastrol: transcriptional signatures of a thiol-targeted molecule.

Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product.

Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells by increasing the expression of bone morphogenetic proteins.

Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

Translational regulation of nuclear gene COX4 expression by mitochondrial content of phosphatidylglycerol and cardiolipin in Saccharomyces cerevisiae.

Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Delta) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5' and 3' untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5' UTR(COX4) revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Delta but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and beta-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Delta cells.

EbpR is important for biofilm formation by activating expression of the endocarditis and biofilm-associated pilus operon (ebpABC) of Enterococcus faecalis OG1RF.

We identify ef1090 (renamed ebpR) and show its importance for the transcriptional regulation of expression of the Enterococcus faecalis pilus operon, ebpABC. An ebpR deletion (DeltaebpR) mutant was found to have reduced ebpABC expression with loss of pilus production and a defect in primary adherence with, as a consequence, reduced biofilm formation.

Enterococcus faecalis rnjB is required for pilin gene expression and biofilm formation.

Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.

RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast.

The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Delta cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the alpha/beta importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathy gene.

OBJECTIVES: We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. BACKGROUND: CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. METHODS: In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. CONCLUSIONS: ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

Reduced vascular endothelial growth factor expression in contusive spinal cord injury.

Vascular endothelial growth factor (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(121) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina.

PURPOSE: To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. METHODS: CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. RESULTS: CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. CONCLUSIONS: The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.

Traumatic brain injury stimulates hippocampal catechol-O-methyl transferase expression in microglia.

Outcome following traumatic brain injury (TBI) is in large part determined by the combined action of multiple processes. In order to better understand the response of the central nervous system to injury, we utilized an antibody array to simultaneously screen 507 proteins for altered expression in the injured hippocampus, a structure critical for memory formation. Array analysis indicated 41 candidate proteins have altered expression levels 24h after TBI. Of particular interest was catechol-O-methyl transferase (COMT), an enzyme involved in metabolizing catecholamines released following neuronal activity. Altered catecholamine signaling has been observed after brain injury, and may contribute to the cognitive dysfunctions and behavioral deficits often experienced after TBI. Our data shows that COMT expression in the injured ipsilateral hippocampus was elevated for at least 14 d after controlled cortical impact injury. We found strong co-localization of COMT immunoreactivity with the microglia marker Iba1 near the injury site. Since dopamine transporter expression has been reported to be down-regulated after brain injury, COMT-mediated catecholamine metabolism may play a more prominent role in terminating catecholamine signaling in injured areas.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

Expression and Regulation of Human Cytochrome P450 4F Isoforms in Tissue Samples and Under TNF-Alpha Challenges

CYP4F (Cytochrome P4504F) enzymes metabolize endogenous molecules including leukotrienes, prostaglandins and arachidonic acid. The involvement of these endogenous compounds in inflammation has led to the hypothesis that changes in the inflamed tissue environment may affect the expression of CYP4Fs during the pro-inflammatory state, which in turn may modulate inflammatory conditions during the anti-inflammatory state. We demonstrated that inflamed tissues have different levels of CYP4F isoform expression profiles in a number of human samples when compared to the average population. The CYP4F isoform expression levels change with the degree of inflammation present in tissue. Further investigation in cell culture studies revealed that inflammatory cytokines, in particular TNF-α, play a role in regulating the expression of the CYP4F family. One of the isoforms, CYP4F11, had different characteristics than that of the other five CYP4F family members. CYP4F11 metabolizes xenobiotics while the other isoforms metabolize endogenous compounds with higher affinity. CYP4F11 also was expressed at high quantities in the brain, and was up-regulated by TNF-α, while the other isoforms were not expressed at high quantities in the brain and were down-regulated by TNF-α. We identified the AP-1 protein of the JNK pathway as the signaling protein that causes significant increase in CYP4F11 expression. Since TNF-α stimulation causes a simultaneous activation of both JNK pathway and NF-κB signaling, we investigated further the role that NF-κB plays on expression of the CYP4F11 gene. We concluded that although there is a significant increase in CYP4F11 expression in the presence of TNF-α, the activation of NF-κB signaling inhibits CYP4F11 expression in a time dependent manner. The expression of CYP4F11 is only significantly increased after 24 hours of treatment with TNF-α; at shorter time points NF-κB signaling overpowers the JNK pathway activation. We believe that these findings may in the future lead to improved drug design for modulating inflammation.