Objective: To define the gene expression patterns during the early phases of a bacterial middle ear infection in the rat model.Method: Using cDNA gene array technology, we profiled the mRNA expression of 1176 genes in a rat model of acute otitis media. We identified changes in gene expression two-fold or greater 12 and 48 h after bilateral ME inoculation with either tryptic soy broth (TSB) or Streptococcus pneumoniae in TSB.Results: Transcripts of cytokines and cell adhesion molecules were up-regulated by 12 h, but returned to placebo transcription levels by 48 h. Three of six stress-response genes, including inducible nitric oxide synthase, GADD45 and heat shock protein 27 (HSP27) were up-regulated by 12 h, with HSP27 transcription levels continuing to rise through 48 h. All assayed transcription factors were up-regulated by 12 h, but only c-fos and c-jun up-regulation persisted to the 48-h time point. Up-regulation of apoptosis-related genes, except for bcl-x, was not evident until 48 h. These gene expression patterns reflected an early proinflammatory response consisting of cytokines, cell adhesion and stress-response molecules at 12 h followed by an up-regulation of apoptosis-related genes at 48 h.Conclusion: Downstream targets of several transcription factors, up-regulated transiently at 12 h, control secondary effects of S. pneumoniae infection, including apoptosis of neutrophils and mucosal epithetial cells, bone proliferation and promotion of leukocyte differentiation. These observations lead to a greater understanding of the early events in the pathogenesis of an AOM episode and highlight therapeutic targets, which may play a roll in the sequelae of AOM. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

The transient receptor potential cation channel subfamily V (TRPV) is a non-specific cation ion channel receptor family that is gated by heat, protons, low extracellular osmolarity and arachidonic acid derivatives. Since some of these endogenous agonists of TRPV receptors are reactive oxygen intermediates produced by lipoxygenases, it has been hypothesized that some members of the TRPV family may respond to challenges by reactive oxygen species. This study used real-time PCR to quantitatively track changes in TRPV1-4 mRNA expression in the spiral, vestibular, and trigeminal ganglia and the kidney from kanamycin (KM)-treated mice. TRPV1, TRPV2, TRPV3 and TRPV4 mRNAs were expressed in spiral and vestibular ganglia, and TRPV2 and TRPV1 mRNAs were most predominant in control mice. After KM (700 mg/kg s.c. b.i.d., 14 days), TRPV1 mRNA and protein expression were significantly up-regulated both in the spiral and vestibular ganglia, but expression was unaffected in the trigeminal ganglion and kidney. Real-time PCR also demonstrated a significant down-regulation in TRPV4 mRNA expression in the inner ear ganglia and kidney after KM treatment. All these mRNA and protein expression changes were eliminated by simultaneous administration of dihydroxybenzoate (300 mg/kg s.c. b.i.d., 14 days), an anti-oxidant that blocks KM ototoxicity. It is proposed that up-regulated TRPV1 expression during KM exposure may promote ganglion cell survival by contributing to neuronal depolarization, with KM-induced tinnitus and dizziness as consequences.

Uncoupling proteins (UCPs) are a proton transporter family located in the mitochondrial inner membrane. The molecular expression and activity of UCPs in brown adipose tissue and skeletal muscle are regulated by factors as diverse as chronic overeating and cold exposure, suggesting roles in energy expenditure and heat production. Although UCP2, UCP4 and brain mitochondrial carrier protein-1 (BMCP-1, i.e. UCP5) mRNAs are expressed in the central nervous system, their central function is unknown. This study presents the first evidence on localization and quantitative expression of UCPs in the rat inner ear by real-time PCR and immunohistochemistry. Real-time PCR studies revealed that UCP2 mRNA was expressed in the vestibular and spiral ganglia more abundantly than any other UCP. Neocortex, by contrast, contained UCP2 and UCP4 equally. Notably, UCP3 and UCP4 mRNAs were expressed in inner ear ganglia, but brain UCP3 mRNA expression level was undetectable by simple PCR. Immunohistochemical studies confirmed that both UCP2- and UCP3-like immunoreactivities were detected in vestibular and spiral ganglion cells and co-localized with a mitochondrial marker, MitoFluorGreen. According to previous reports, UCP2 and UCP3 are thermogenic in yeast and brain UCP2 has been suggested to modulate pre- and post-synaptic events by axonal thermogenesis. It has also been reported recently that UCP2 and UCP3 responses to superoxide application may be an antioxidant protective mechanism. Therefore, it is suggested that mitochondrial UCPs (UCP2, UCP3, UCP4) may play both a protective role against oxidative damage and a thermal signaling role in the eighth nerve.

The type 1 vanilloid receptor (VR1) is a non-specific cation channel activated by capsaicin, lipoxygenase (LOX) products, heat and acid. This study demonstrates VR1 and 5-LOX expression by inner ear ganglion cells. A PCR product (210 bp) was amplified from both oligo(dT)- and random primer-generated cDNAs of rat spiral ganglion cells using VR1 gene-specific primers constructed from the 3′ non-homologous region. This PCR product shared 100% sequence homology to a rat VR1 cDNA (GenBank accession no. AF029310) and a rat vanilloid receptor splice variant mRNA (GenBank accession no. AF158248). Frozen sections of PLP-fixed, decalcified Long–Evans rat temporal bones were stained immunohistochemically for VR1. Neurons and satellite cells in both the vestibular and spiral ganglia were VR1-immunopositive. Neurons and supporting cells in adjacent sections of these ganglia were immunopositive for 5-LOX. These findings raise the hypothesis that activation of VR1 by endogenous ligands may contribute to hypersensitivity of the eighth nerve to hair cell inputs in a variety of pathologic conditions, such as tinnitus, Meniere’s disease and migraine. In particular, these data suggest that LOX activation during inflammatory processes or during cyclo-oxygenase inhibition (e.g. by aspirin) is a potential intrinsic source of VR1 activation in inner ear ganglia.

Our laboratory has employed replication-defective herpes simplex virus type 1 gene transfer vectors for treatment of animal models of human malignant glioblastoma. The base vectors were defective for the immediate early (IE) genes ICP4, ICP27, and ICP22 but expressed the IE gene ICP0, which can arrest tumor cell division, and an IE thymidine kinase (α-tk) gene construct that mediates suicide gene therapy (SGT) in the presence of ganciclovir (GCV). Previously, we reported that SGT using ICP0/α-tk vectors in nude mouse models of glioblastoma was improved by coexpression of the gap-junction-forming protein connexin43 (Cx43) or human tumor necrosis factor α (TNFα). We also showed that further gains in therapeutic outcome could be achieved by combining TNFα-enhanced SGT with gamma-knife radiosurgery (GKR). To expand these observations, we have first repeated these studies in immunocompetent rats with brain tumors derived from implanted 9L gliosarcoma cells and second compared the most efficient vector from this study with a new recombinant vector, NUREL-C2, which expressed both TNFα and Cx43 along with ICP0 and α-tk. Results from the first part indicated that our ICP0/α-tk/TNFα vector in combination with GKR provides an effective therapy although this treatment was not statistically better than GKR combined with the ICP0/α-tk/Cx43 vector. Our observations in the second part suggested that NUREL-C2 may be more effective than the ICP0/α-tk/TNFα vector in combination treatments with GCV (P = 0.08) or GCV plus GKR (P = 0.10). GKR significantly enhanced the efficacy of NUREL-C2/GCV treatment (P = 0.02) as well as other virus/GCV treatments (P ≤ 0.05). Conversely, the efficacy of GKR was significantly improved by both the ICP0/α-tk/TNFα vector and NUREL-C2 in combination with GCV (P = 0.02 and P < 0.01, respectively). Together these results indicate that NUREL-C2 may be an attractive candidate for Phase I gene-therapy safety studies in patients with recurrent malignant glioblastoma.

The RING domain protein Sina, together with Phyllopod and the F-box protein Ebi, forms a Ras-regulated E3 ubiquitin ligase complex that activates photoreceptor cell differentiation in the eye of Drosophila melanogaster. The expression of Phyllopod is induced upon Ras activation, allowing the complex to degrade the transcription repressor Tramtrack and removing its block of neuronal development in photoreceptor precursors. We show that Phyllopod functions as an adaptor in the complex, physically linking Sina with Tramtrack via separate binding domains. One 19-amino-acid domain in Phyllopod interacts with a region of Sina's SBD domain. Another domain in Phyllopod interacts with a C-terminal helix in the POZ domain of Tramtrack. This interaction is specific to the Tramtrack POZ domain and not to other POZ domain proteins present in photoreceptor precursors. Degradation of Tramtrack is dependent upon association of Sina with its cognate binding site in Phyllopod. These results illustrate how Ras signaling can modulate an E3 ligase activity not by the phosphorylation of substrate proteins but by regulating the expression of specific E3 adaptors.

INTRODUCTION:Chronic alcohol consumption predisposes susceptible individuals to both acute and chronic pancreatitis.AIMS:Our hypothesis was that alcohol increases the risk of pancreatitis by disrupting defense mechanisms and/or enhancing injury-associated pathways through altered gene expression. Hence, we studied the expression of pancreatic genes in rats chronically exposed to ethanol.METHODOLOGY:Male Wistar rats were pair-fed liquid diets without and with ethanol for 4 weeks. Total RNA was extracted from rat pancreas and other organs. The mRNA expression patterns among pancreatic samples from ethanol-fed rats and controls were compared with use of mRNA differential display. The differentially expressed cDNA tags were isolated, cloned, and sequenced.RESULTS:One cDNA tag that was overexpressed in the pancreas showed 99% sequence homology to a rat pancreatic cholesterol esterase mRNA (CEL; Enzyme Commission number [EC] 3.1.1.13). The differential expression was confirmed by realtime PCR. Gene expression was also increased in the liver but not in the heart or brain of the alcohol-fed rats. Because CEL has fatty acid ethyl ester (FAEE)-generating activity and FAEEs play a major role in acute alcoholic pancreatitis, we determined the expression of other genes encoding for FAEE-generating enzymes and showed similar organ-specific expression patterns.CONCLUSION:Our results demonstrate that chronic ethanol consumption induced expression of FAEE-related genes in the pancreas and liver. This upregulation may be a central mechanism leading to acinar cell injury.

Sex differences in susceptibility to alcohol-induced liver injury have been observed in both humans and experimental animal models. Using a standard model of alcohol-induced fatty liver injury and microarray analysis, we have identified differential expression of hepatic genes in both sexes. The genes that exhibit differential expression are of three types: those that are changed only in male rats fed alcohol, those that change in only female rats fed alcohol, and those that change in both sexes, although not always in the same manner. Certain of the differentially expressed genes have previously been identified as participants in the induction of alcohol-induced liver injury. However, this analysis has identified a number of genes that heretofore have not been implicated in alcoholic liver injury; such genes may provide new areas of investigation into the pathogenesis of this disease.

expression of the Notch signaling cascade during AP.METHODS.AP was induced in female Swiss Webster mice by 8 hourly injections of caerulein (100 ~g/kg BW/injection, IP).Mice were sacrificed at regular intervals and pancreatic poly (A +) RNA prepared.Pancreatic expression of Notch-l, HES, Delta-like gene 1 (DIll) and RBP-Jk 1 were examined by Northern blotting analyses.RESULTS.Pancreatic expression of Notch-l, DIll (data not shown), RBP-Jk and HES increased significantly in a temporally dependent fashion.Maximal expression of the Notch signaling pathway occurred during the regenerative phase of AP (ie, acinar cell replacement) and was coordinated with pancreatic cell proliferation.CONCLUSIONS.Our findings show that the Notch signaling cascade is activated during AP and acinar cell replacement.The increased expression of the Notch signaling cascade during the proliferative phase of acinar cells during AP suggests that it is important in guiding pancreatic progenitor cells in their choice between uninterrupted proliferation or acinar cell differentiation. Pancreatic expression of theNotch signaling cascade after induc~ of AP Oh 8h 12h 245 48h 96h 14d

Individuals with chronic excessive alcohol ingestion are put at the risk of acute and chronic pancreatitis. Underlying molecular mechanisms are unknown. Differential gene expression in the pancreas was profiled using mRNA differential display by comparison between control and ethanol-consuming rats. Male Wistar rats were fed with diets containing 6.7% (vol/vol) ethanol for 4 wk. A cDNA tag that was overexpressed in the pancreas of rats fed ethanol was isolated. A 723-bp cDNA was cloned from a rat pancreatic cDNA library, which encodes a novel rat mitochondrial ATP synthase subunit 9, isoform 3 (ATP5G3), which is homologous to a human ATP5G3 gene. Real-time PCR demonstrated that all three nuclear gene isoforms (ATP5G1, ATP5G2, and ATP5G3) were consistently upregulated in the pancreas of alcohol-consuming rats, parallel with mitochondrial injury. The cellular response to mitochondrial damage and metabolic stress may reflect an adaptive process for mitochondrial repair in pancreatic acinar cells during chronic ethanol ingestion.

Background/Purpose: Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. Methods: Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. Results: Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the η subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. Conclusions: With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient α-smooth muscle actin to form the stress fibers characteristic of these cells. J Pediatr Surg 35:406-419. Copyright © 2000 by W.B. Saunders Company.

BACKGROUND:Scar formation and subglottic stenosis often cause health problems in surgical otolaryngology. However, fetal wounds demonstrate scarless healing. The underlying mechanism remains poorly understood. We isolated differentially expressed genes by comparison between nonwounded with wounded skin of fetal and adult rabbits.METHODS:Skin incisional wounds were made in fetal (21 to 23 days' gestation) and adult rabbits. Nonwounded and wounded skin were harvested 12 hours after surgery. Total RNA was extracted. By means of messenger RNA differential display, differentially expressed complementary DNA fragments were isolated, cloned, and sequenced. The expressed transcripts were verified by reverse RNA dot blot and semiquantitative reverse transcription and polymerase chain reaction.RESULTS:One complementary DNA tag that was induced in fetal skin wounds and repressed in adult skin wounds was isolated. The sequence of this complementary DNA (352 base pairs) encodes the messenger RNA for the E-prostanoid (EP) 4 receptor for prostaglandin E(2) (PGE(2)). The truly differential expression of the transcript was confirmed. In normal skin, the EP4 receptor messenger RNA levels were higher in adults than in fetuses. Twelve hours after wounding, the EP4 receptor transcript was remarkably induced in fetal skin wounds but repressed in adult skin wounds.CONCLUSIONS:Our study demonstrates the differential expression of the EP4 receptor messenger RNA in fetal and adult skin before and 12 hours after wounding. Our results suggest that prostaglandin E(2) is involved in the differential cellular responses and in the regulation of the intracellular signal transduction through its binding to EP4 receptor during fetal wound repair.

ebi regulates the epidermal growth factor receptor (EGFR) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes EGFR-dependent down-regulation of Tramtrack88, an antagonist of neuronal development.