Ha-Sheng Li

Pancreatic cholesterol esterase, ES-10, and fatty acid ethyl ester synthase III gene expression are increased in the pancreas and liver but not in the brain or heart with long-term ethanol feeding in rats.

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.

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

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 eta 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 alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Suppression of Epithelial Ion Transport Transcripts during Pneumococcal Acute Otitis Media in the Rat

Until recently, it was not feasible to conduct genome-wide screening for gene transcript variations that play key roles in the pathogenesis of otitis media. In this study microarray technology was used to profile differential gene expression patterns from rat middle ear mucosa at 12 and 48 h after Streptococcus pneumoniae challenge. Real-time polymerase chain reaction was performed for independent verification of the microarray results. Three ion transport mRNAs were simultaneously suppressed more than 4-fold at 12 h in bacteria-challenged ears, including Na,K-ATPase alpha 1 subunit (SPATPa1), sodium channel beta 2 subunit (SCNB2) and sodium -hydrogen exchange protein isoform 2 subunit (NHE2). At 48 h after infection, the mRNA levels of SCNB2 and NHE2 had decreased 7- and 10-fold, respectively, whereas the relatively abundant SPATPa1 transcript showed recovery. The downregulation of Na + -transporting transcripts suggests a reduced number of epithelial cells and transporting proteins and/or the dysfunction of sodium transporters secondary to the bacterial infection. These changes can disrupt the coupling of the apical Na + entry and basolateral Na + extrusion, deplete the electrochemical Na + transmembrane gradient, disrupt the intracellular osmotic equilibrium and lead to intracellular acidification and the accumulation of excess sodium, water and other organic and inorganic molecules in the middle ear cavity. Any or all of these changes may contribute to the initiation and persistence of middle ear mucosa inflammation and effusion during an episode of bacterial acute otitis media.

Chronic ethanol consumption induces gene expression of pancreatic monitor peptide, but not SPINK1/PSTI-56, in rats.

The primary factors that predispose humans to the development of alcoholic pancreatitis are unknown. One of the earliest observations in humans in whom this disease develops is pancreatic hypersecretion caused by unknown mechanisms. Messenger RNA (mRNA) differential display was performed in a rat model to investigate the molecular mechanisms associated with ethanol-induced pancreatic hypersecretion. Male Wistar rats were pair-fed Lieber-DeCarli diets with or without ethanol for 7 days or 4 weeks. Total RNA was extracted from the pancreas and its neurohormonal control sites. Differentially expressed complementary DNA (cDNA) tags were isolated, cloned, and sequenced. One 248-bp cDNA was consistently and strongly induced in the pancreata of rats fed ethanol for 4 weeks. The sequence was highly homologous to both rat pancreatic monitor peptide (MP) and pancreatic secretory trypsin inhibitor (PSTI-56), also known as serine protease inhibitor, Kazal type 1 (SPINK1). Confirmatory reverse-transcription–polymerase chain reaction showed that PSTI-56 expression remained unchanged, whereas MP mRNA levels were elevated more than four times in the pancreata of ethanol-fed rats. These results indicate that long-term ethanol ingestion increases MP mRNA levels in the rat pancreas. Because MP stimulates cholecystokinin release and cholecystokinin is an important stimulant of pancreatic secretion, the enhanced MP gene expression may contribute to pancreatic hypersecretion.

Mucosal expression of genes encoding possible upstream regulators of Na+ transport during pneumococcal otitis media.

Objective --Recently, we reported that gene transcripts encoding 3 Na+ transport proteins (pump, channel and exchanger) in the middle ear mucosa (MEM) were simultaneously suppressed at 12 and 48 h after Streptococcus pneumoniae (SP) challenge of rat middle ears. Material and Methods --From cDNA microarray screening of those specimens, several gene clusters, including Nos2 and the transcription factors Fos, Fosl1, Jun and Nfkb1, were identified as possible upstream regulators of Na+ transport protein expression. The altered expression of those genes in MEM was validated and quantified using real-time polymerase chain reaction and MEM protein expression for Atp1a1, Nos2 and Nfkb1 was studied using Western blot and/or immunohistochemistry assays. Results --At both time-points. Atp1a1 mRNA and protein were decreased and Nos2 mRNA and protein were increased in MEM. While Nfkb1 protein was decreased at those times. the corresponding mRNA was increased at 12 h but decreased at 48 h. Gene expression for Fos was suppressed at both times, while that for Fosl1 and Jun was augmented at 12 h and suppressed at 48 h. Immunohistochemical study of specimens challenged with SP showed a swollen MEM with infiltration of inflammatory cells that stained positive for Nos2. Conclusion --Given the known activities of Nos2, these results can be interpreted as evidencing a transcriptional suppression of Na+ transport protein synthesis secondary to upregulated Nos2 expression during SP infection of the rat MEM. This proposed signaling pathway does not require the continuous upregulation of Nfkb1 or the other assayed transcription factors as early as 12 h after middle ear infection.