Identification of Chicken Transglutaminase 1 and In Situ Localization of Transglutaminase Activity in Avian Skin and Esophagus.

Transglutaminase 1 (TGM1) is a membrane-anchored enzyme that cross-links proteins during terminal differentiation of epidermal and esophageal keratinocytes in mammals. The current genome assembly of the chicken, which is a major model for avian skin biology, does not include an annotated region corresponding to TGM1. To close this gap of knowledge about the genetic control of avian cornification, we analyzed RNA-sequencing reads from organotypic chicken skin and identified TGM1 mRNA. By RT-PCR, we demonstrated that TGM1 is expressed in the skin and esophagus of chickens. The cysteine-rich sequence motif required for palmitoylation and membrane anchorage is conserved in the chicken TGM1 protein, and differentiated chicken keratinocytes display membrane-associated transglutaminase activity. Expression of TGM1 and prominent transglutaminase activity in the esophageal epithelium was also demonstrated in the zebra finch. Altogether, the results of this study indicate that TGM1 is conserved among birds and suggest that chicken keratinocytes may be a useful model for the study of TGM1 in non-mammalian cornification.

Obesity alters Ace2 and Tmprss2 expression in lung, trachea, and esophagus in a sex-dependent manner: Implications for COVID-19.

Obesity is a major risk factor for SARS-CoV-2 infection and COVID-19 severity. The underlying basis of this association is likely complex in nature. The host-cell receptor angiotensin converting enzyme 2 (ACE2) and the type II transmembrane serine protease (TMPRSS2) are important for viral cell entry. It is unclear whether obesity alters expression of Ace2 and Tmprss2 in the lower respiratory tract. Here, we show that: 1) Ace2 expression is elevated in the lung and trachea of diet-induced obese male mice and reduced in the esophagus of obese female mice relative to lean controls; 2) Tmprss2 expression is increased in the trachea of obese male mice but reduced in the lung and elevated in the trachea of obese female mice relative to lean controls; 3) in chow-fed lean mice, females have higher expression of Ace2 in the lung and esophagus as well as higher Tmprss2 expression in the lung but lower expression in the trachea compared to males; and 4) in diet-induced obese mice, males have higher expression of Ace2 in the trachea and higher expression of Tmprss2 in the lung compared to females, whereas females have higher expression of Tmprss2 in the trachea relative to males. Our data indicate diet- and sex-dependent modulation of Ace2 and Tmprss2 expression in the lower respiratory tract and esophagus. Given the high prevalence of obesity worldwide and a sex-biased mortality rate, we discuss the implications and relevance of our results for COVID-19.

Impact of Deoxycholic Acid on Oesophageal Adenocarcinoma Invasion: Effect on Matrix Metalloproteinases.

Bile acids (BAs) have been implicated in the development of oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma (OAC). However, whether BAs promote cancer invasiveness has not been elucidated. We evaluated the role of BAs, in particular deoxycholic acid (DCA), in OAC invasion. Migration and invasiveness in untreated and BA-treated oesophageal SKGT-4 cancer cells were evaluated. Activity and expression of different matrix metalloproteinases (MMPs) were determined by zymography, ELISA, PCR and Western blot. Finally, human OAC tissues were stained for MMP-10 by immunohistochemistry. It was found that SKGT-4 cells incubated with low concentrations of DCA had a significant increase in invasion. In addition, MMP-10 mRNA and protein expression were also increased in the presence of DCA. MMP-10 was found to be highly expressed both in-vitro and in-vivo in neoplastic OAC cells relative to non-neoplastic squamous epithelial cells. Our results show that DCA promotes OAC invasion and MMP-10 overexpression. This study will advance our understanding of the pathophysiological mechanisms involved in human OAC and shows promise for the development of new therapeutic strategies.

Expression of metalloproteinase-9 in patients with mild and severe forms of gastroesophageal reflux disease.

BACKGROUND: Gastroesophageal reflux disease develops when the stomach contents causes troublesome symptoms and complications. Mild forms are non-erosive and erosive esophagitis, and severe forms are Barrett's esophagus and Esophageal adenocarcinoma. Matrix metalloproteinases are endopeptidases that can degrade components of the extracellular matrix, they play an important role in tumor invasion as well as in metastasis. OBJECTIVE: To correlate the expression of metalloproteinase 9 (MMP-9) in esophageal biopsies from patients with mild and severe forms of Gastroesophageal reflux disease. METHOD: Cross-sectional study. The expression of MMP-9 was determined in biopsies of esophageal tissue of patients with mild and severe GRD. The included variables were age, sex, diagnosis, smoking and alcoholic habits, body mass index (BMI) and expression of MMP-9. Descriptive statistics was performed, Kappa for concordance in diagnosis as well as X2. RESULTS: There were 50 patients, 32 (64%) men and 18 (36%) women, mean age 52.13 ± 14.75 years of age. 12 (24%) with smoking and 7 (14%) with alcoholism. Average BMI was 26.71 ± 4.07 kg/m2 (15 to 33); 40 (80%) with obesity. The inter observer concordance for histopathological diagnosis was 1.0 and 0.84 for esophagitis degrees. 27 (54%) patients had esophagitis, 16 (32%) Barrett's esophagus and 7 (14%) esophageal cancer. There was expression of MMP-9 in four patients with esophagitis, five with Barrett's esophagus and five with esophageal cancer. Statistical significance was found between the expression of MMP-9 and smoking (p = 0.011) and histopathological diagnosis (p = 0.052). CONCLUSIONS: The expression of MMP-9 is most common in severe forms compared to the mild forms of GRD.

ANTECEDENTES: La enfermedad por reflujo gastroesofágico (ERGE) se desarrolla cuando el contenido estomacal ocasiona síntomas molestos o complicaciones. Las formas leves son esofagitis no erosiva y erosiva; las graves, esófago de Barrett y adenocarcinoma esofágico. Las metaloproteinasas de la matriz degradan componentes de la matriz extracelular, y tienen un papel importante en la invasión tumoral y la metástasis. OBJETIVO: Relacionar la expresión de la metaloproteinasa-9 (MMP-9) en biopsias esofágicas de pacientes con formas leves y graves de ERGE. MÉTODO: Estudio transversal. Se determinó la expresión de MMP-9 en biopsias esofágicas de pacientes con ERGE grave y leve. Las variables fueron edad, sexo, diagnóstico, tabaquismo, alcoholismo, índice de masa corporal (IMC) y expresión de MMP-9. Se realizó estadística descriptiva, concordancia para el diagnóstico y prueba de ji al cuadrado. RESULTADOS: 50 pacientes, 32 (64%) hombres y 18 (36%) mujeres, con edad media de 52.13 ± 14.75 años. Doce (24%) fumadores y 7 (14%) con alcoholismo. El IMC promedio fue de 26.71 ± 4.07 kg/m2 (rango: 15-33); 40 (80%) eran obesos. La concordancia entre observadores para el diagnóstico histopatológico fue de 1.0, y de 0.84 para esofagitis. Veintisiete (54%) tuvieron esofagitis, 16 (32%) esófago de Barrett y 7 (14%) cáncer de esófago. Hubo expresión de MMP-9 en cuatro pacientes con esofagitis, cinco con esófago de Barrett y cinco con cáncer esofágico. Encontramos diferencia estadísticamente significativa entre la expresión de MMP-9 y el tabaquismo (p = 0.011) y el diagnóstico histopatológico (p = 0.052). CONCLUSIONES: La expresión de MMP-9 es más frecuente en las formas graves que en las leves de ERGE.

Characterization of a Schistosoma mansoni NDPK expressed in sexual and digestive organs.

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.

Thymidine phosphorylase and angiogenesis in early stage esophageal squamous cell carcinoma.

BACKGROUND: The relationship between thymidine phosphorylase (TP) and angiogenesis at the early stage of esophageal squamous cell carcinoma has been unclear. METHODS: Using 14 samples of normal squamous epithelium, 11 samples of low-grade intraepithelial neoplasia, and 64 samples of superficial esophageal cancer, microvessel density (MVD) was estimated using immunostaining for CD34 and CD105. TP expression was also evaluated in both cancer cells and stromal monocytic cells (SMCs). We then investigated the correlation between MVD and TP expression in both cancer cells and SMCs. RESULTS: On the basis of the above parameters, MVD was significantly higher in cancerous lesions than in normal squamous epithelium. In terms of CD34 and CD105 expression, MVD showed a gradual increase from normal squamous epithelium, to low-grade intraepithelial neoplasia, and then to M1 and M2 cancer, and M3 or deeper cancer. M1 and M2 cancer showed overexpression of TP in both cancer cells and SMCs. There was no significant correlation between TP expression in cancer cells and MVD estimated from CD34 (rS = 0.16, P = 0.21) or CD105 (rS = 0.05, P = 0.68) expression. Significant correlations were found between TP expression in SMCs and CD34-related (rS = 0.46, P < 0.001) and CD105-related (rS = 0.34, P < 0.01) MVD. In M3 or deeper cancers, there were no significant correlations between TP expression in cancer cells or SMCs and venous invasion, lymphatic invasion, and lymph node metastasis. CONCLUSION: TP expression is activated in both cancer cells and stromal monocytic cells at the very early stage of ESCC progression. TP expression in SMCs, rather than in cancer cells, is significantly correlated with angiogenesis.

Dysregulation of PARP1 is involved in development of Barrett's esophagus.

AIM: To investigate the potential role of poly(ADP-ribose) polymerase 1 (PARP1) in the development of Barrett's esophagus (BE). METHODS: A BE mouse model was established to examine the esophageal morphological changes and molecular changes. Microarray analysis was performed to compare the gene expression profiles between BE patients and healthy controls. qPCR was used to examine the PARP1 expression in cell lines after treatment with H2O2 and bile acids (pH 4). Immunofluorescence staining, comet assay, and annexin V staining were used to evaluate the impact of PARP1 activity on cell survival and DNA damage response after oxidative stress. RESULTS: The gene expression profile in normal and BE esophageal epithelial cells showed that PARP1, the major poly(ADP-ribose) polymerase, was overexpressed in BE. In the mouse model of BE, positive staining for NF-κB, γH2AX, and poly(ADP-ribose) (PAR) was observed. H2O2 and bile acids (pH 4) increased the PARP1 mRNA expression level in normal esophageal epithelial cells. Using shRNA-PARP1 to suppress PARP1 activity decreased the cell viability after treatment with H2O2 and bile acids (pH 4), and increased the oxidative damage as demonstrated by an increase in the levels of H2O2, intracellular reactive oxygen species (ROS), oxidative DNA damage, double-strand breaks, and apoptosis (P < 0.01). CONCLUSION: The dysfunction of PARP1 in esophageal epithelial cells increases the levels of ROS and oxidative DNA damage, which could be common risk factors for BE and esophageal adenocarcinoma.

ADAM10 mediates the cell invasion and metastasis of human esophageal squamous cell carcinoma via regulation of E-cadherin activity.

A disintegrin and metalloprotease 10 (ADAM10) is involved in the tumorigenesis, invasion and metastasis of several types of solid tumors. However, the potential role of ADAM10 in human esophageal squamous cell carcinoma (ESCC) is not yet well understood. The present study showed that ADAM10 was overexpressed in human ESCC tissues in vivo, and positively associated with depth of tumor invasion, lymph node metastasis and TNM stage, contributing to tumor carcinogenesis, invasion and metastasis. Additionally, ADAM10 was overexpressed in 3 types of ESCC cell lines in vitro, as compared to that in normal esophageal epithelial cells (NEECs); and moreover, ESCC cells with high ADAM10 expression obtained enhanced invasion and migration ability. Subsequently, ADAM10 silencing by small interfering (si) RNA in ESCC cell line, EC-1, reduced cell invasion, migration and proliferation in vitro. Finally, ADAM10 negatively regulated E-cadherin in ESCC in vivo and in vitro. In conclusion, active ADAM10 promotes the carcinogenesis, invasion, metastasis and proliferation of ESCC and controls invasion and metastasis at least in part through the shedding of E-cadherin activity, which makes it a potential biomarker and a useful therapeutic target for ESCC.

Hydrogen sulfide synthesis enzymes reduced in lower esophageal sphincter of patients with achalasia.

The etiology of achalasia remains largely unknown. Considerable evidence reveals that the lower esophageal sphincter dysfunction is due to the lack of inhibitory neurotransmitter, secondary to esophageal neuronal inflammation or loss. Recent studies suggest hydrogen sulfide may act as an inhibitory transmitter in gastrointestinal tract, but study about hydrogen sulfide in human esophagus still lack. The aim of the study was to investigate if hydrogen sulfide synthesis enzymes could be detected in human esophagus and if the synthesis of the endogenous hydrogen sulfide could be affected in achalasia patients. Tissue samples in cardia, lower esophageal sphincter, 2 cm and 4 cm above lower esophageal sphincter were obtained from achalasia patients undergoing peroral endoscopic myotomy. Control tissues in lower esophageal sphincter were obtained from esophageal carcinoma patients. Expression of cystathionine-ß-synthase and cystathionine-γ-lyase in lower esophageal sphincter of achalasia patients and control were detected by immunohistochemical staining. In addition, expression of cystathionine-ß-synthase and cystathionine-γ-lyase were compared among different parts of esophagus in achalasia patients. Compared with control, the expression of cystathionine-ß-synthase and cystathionine-γ-lyase in lower esophageal sphincter of achalasia patients was significantly reduced (χ2 = 11.429, P = 0.010). The expression of cystathionine-ß-synthase and cystathionine-γ-lyase were lower in lower esophageal sphincter than that in 2 cm and 4 cm above lower esophageal sphincter, respectively (all P < 0.05). In conclusion, the expression of hydrogen sulfide synthesis enzymes, cystathionine-ß-synthase and cystathionine-γ-lyase, can be detected in human esophagus and is reduced in patients with achalasia, which implicates the involvement of the two hydrogen sulfide synthesis enzymes in the pathophysiology of achalasia.

The Schistosome Esophagus Is a 'Hotspot' for Microexon and Lysosomal Hydrolase Gene Expression: Implications for Blood Processing.

BACKGROUND: The schistosome esophagus is divided into anterior and posterior compartments, each surrounded by a dense cluster of gland cell bodies, the source of distinct secretory vesicles discharged into the lumen to initiate the processing of ingested blood. Erythrocytes are lysed in the lumen, leucocytes are tethered and killed and platelets are eliminated. We know little about the proteins secreted from the two glands that mediate these biological processes. METHODOLOGY/PRINCIPAL FINDINGS: We have used subtractive RNA-Seq to characterise the complement of genes that are differentially expressed in a head preparation, compared to matched tissues from worm tails. The expression site of representative highlighted genes was then validated using whole munt in situ hybridisation (WISH). Mapping of transcript reads to the S. mansoni genome assembly using Cufflinks identified ~90 genes that were differentially expressed >fourfold in the head preparation; ~50 novel transcripts were also identified by de novo assembly using Trinity. The largest subset (27) of secreted proteins was encoded by microexon genes (MEGs), the most intense focus identified to date. Expression of three (MEGs 12, 16, 17) was confirmed in the anterior gland and five (MEGs 8.1, 9, 11, 15 and 22) in the posterior gland. The other major subset comprised nine lysosomal hydrolases (aspartyl proteases, phospholipases and palmitoyl thioesterase), again localised to the glands. CONCLUSIONS: A proportion of the MEG-encoded secretory proteins can be classified by their primary structure. We have suggested testable hypotheses about how they might function, in conjunction with the lysosomal hydrolases, to mediate the biological processes that occur in the esophagus lumen. Antibodies bind to the esophageal secretions in both permissive and self-curing hosts, suggesting that the proteins represent a novel panel of untested vaccine candidates. A second major task is to identify which of them can serve as immune targets.

Inhibition of p38 MAPK activation attenuates esophageal mucosal damage in a chronic model of reflux esophagitis.

BACKGROUND: Reflux esophagitis (RE) is one of the common gastrointestinal diseases that are increasingly recognized as a significant health problem. This study was designed to investigate the role of p38 mitogen-activated protein kinase (MAPK) in experimental chronic RE model of rats. METHODS: Chronic acid RE rats were induced by fundus ligation and partial obstruction of the pylorus and treated with SB203580 (a p38 MAPK inhibitor, i.p., 1 mg/kg/day) for 14 days. KEY RESULTS: Immunohistochemical staining and Western blotting results revealed the activation of p38 MAPK signaling in the esophagus mucosa 14 days post injury. Through gross and histological assessment, we found that inhibition of p38 MAPK activation by SB203580 attenuated esophageal mucosal damage in RE rats. Inhibition of p38 MAPK activation in RE rats attenuated esophageal barrier dysfunction, through enhancing the expression of tight junction proteins and reducing the expression of matrix matalloproteinases-3 and -9. Inhibition of p38 MAPK activation in RE rats reduced CD68-positive cells in esophagus mucosa and mRNA levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß in esophagus and protein levels of TNF-α, IL-6, and IL-1ß in serum. In addition, we found that inhibition of p38 MAPK activation in RE rats suppressed protein expression of inducible nitric oxide synthase and reduced formation of nitric oxide (NO), 3-nitrotyrosin, and malondialdehyde in esophagus. CONCLUSIONS & INFERENCES: Inhibition of p38 MAPK activation attenuated esophageal mucosal damage in acid RE rats, possibly by modulating esophageal barrier function and regulating inflammatory cell recruitment, and the subsequent formation of cytokines, NO, and reactive oxygen species.

PCSK5 mutation in a patient with the VACTERL association.

BACKGROUND: The VACTERL association is a typically sporadic, non-random collection of congenital anomalies that includes vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula with esophageal atresia, renal anomalies, and limb abnormalities. Although several chromosomal aberrations and gene mutations have been reported as disease-causative, these findings have been sparsely replicated to date. CASE PRESENTATION: In the present study, whole exome sequencing of a case with the VACTERL association uncovered a novel frameshift mutation in the PCSK5 gene, which has been reported as one of the causative genes for the VACTERL association. Although this mutation appears potentially pathogenic in its functional aspects, it was also carried by the healthy father. Furthermore, a database survey revealed several other deleterious variants in the PCSK5 gene in the general population. CONCLUSIONS: Further studies are necessary to clarify the etiological role of the PCSK5 mutation in the VACTERL association.

Esophageal COX-2 expression is increased in Barrett's esophagus, obesity, and smoking.

BACKGROUND: Increased esophageal cyclooxygenase-2 (COX-2) expression has been associated with Barrett's esophagus (BE); however, it is unknown whether COX-2 expression varies among patient groups with different clinical or socio-demographic factors. METHODS: We conducted a case-control study among eligible patients scheduled for elective esophagogastroduodenoscopy and patients eligible for screening colonoscopy recruited from primary clinics. We compared 39 BE tissue samples and 47 squamous tissue samples from BE cases and 240 squamous tissue samples from controls. Clinical and socio-demographic data were prospectively collected. Immunohistochemical staining for esophageal COX-2 was performed and scored. RESULTS: The median COX-2 score was significantly higher in BE tissue than squamous tissue from cases or controls (p < 0.001). Median COX-2 expression levels were higher in tissue samples from participants with a waist-to-hip ratio (WHR) in the 2nd tertile [unadjusted odds ratio (OR) 2.04; 95 % confidence interval (95 % CI) 1.17-3.57] and 3rd tertile (unadjusted OR 2.24; 95 % CI 1.20-4.16) compared with the 1st tertile and from current smokers compared with former or non-smokers (unadjusted OR 1.68; 95 % CI 1.03-2.75). In the multivariate analysis, WHRs in the 2nd tertile (OR 1.92; 95 % CI 1.07-3.45) and the 3rd tertile (OR 2.14; 95 % CI 1.10-4.16) were associated with high COX-2 compared with the 1st tertile, as was current smoking (OR 1.78; 95 % CI 1.06-2.97) compared with former and non-smoking. CONCLUSION: We found a significant association between elevated esophageal mucosa COX-2 levels and the presence of BE tissue, as well as between elevated COX-2 levels and high WHR and current tobacco smoking. This information may assist in identifying patients likely to benefit from chemoprevention with COX-2 inhibitors.

[FUNCTIONING PROTEASES IN THE ESOPHAGUS MUCOSA AFTER CHEMICAL BURNS].

The main result of esophagus burn is the formation of scars, that caused by excessive synthesis of collagen and changes the balance of metalloproteinases and their tissue inhibitors. It was studied the activity of proteolytic enzymes, participation of MMP (metalloproteinase) and their tissue inhibitors (TIMP) in alkali burns of the esophagus 1st and 2nd degrees. We have shown a significant increase of TIMP level in homogenate after alkali burns of the esophagus (an average of 31-56% depend on of burn degree). We observed a reduced activity of serine proteinase after 1st degree burns on 15th, 21st day 35 and 18% respectively, after burns 2nd degree on 15th, 21st day 54 and 50%. The decrease of activity MMP after 1st degree burns on 15th and 21st day 30, 19%, respectively, in conditions of chemical burns 2nd degree on 15th and 21st day 30, 37%. These data may indicate the development of scarring after burn simulation of 2nd degree. Further investigation of the MMP and TIMP in the process of wound healing can be useful in creating effective approaches to prevent formation of post scarring of the esophagus.

Activity of mitogen-activated protein kinases in the esophageal epithelium of patients with Barrett's esophagus.

Barrett's esophagus (BE), a complication of gastroesophageal reflux disease, is associated with an increased risk of esophageal cancer. Mitogen-activated protein kinases may play an important role in the pathogenesis of this process. We aimed to evaluate mitogen-activated protein kinases activity in esophageal mucosa of patients with BE and find possible relationship between reflux type and BE. Twenty-four patients (mean age: 59 years) with gastroesophageal reflux disease symptoms and endoscopically suspected esophageal metaplasia (ESEM) were prospectively enrolled for testing by a multichannel intraluminal impedance monitoring along with a Bilitec 2000. Endoscopic biopsies were taken from methylene blue-positive pit patterns (sites suggesting specialized intestinal metaplasia [SIM]), from 2 cm above the Z-line and from cardial parts of the stomach. The biopsies were analyzed for extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 activity by Western blot. Seventeen ESEMs had histologically proven metaplasia: eight patients had SIM and nine had gastric-type epithelia (GE). Biliary reflux was more evident in SIM (P = 0.019) but not in GE (P = 0.019); non-biliary reflux was typical for GE (P = 0.005) but not for SIM (P = 0.04). Strong activations of ERK and p38 were found predominantly in SIM, but not in normal esophageal mucosa (NE) (P = 0.01 and P < 0.001 respectively). Strong signals for active JNK and p38 were detected in GE, but not in NE (P = 0.006 and P = 0.02 respectively). ERK activity was significantly higher than p38 activity in ESEM patients only with GE (P = 0.02). The strong activation of ERK, but not JNK is indicative of SIM. The presence of bile in gastroesophageal refluxate is predisposing to SIM, but not to GE in esophageal mucosa.

Increased immunoreactivity of cathepsins in the rat esophagus under chronic acid reflux esophagitis.

We have designed a stable rat chronic acid reflux esophagitis (RE) model. In gastrointestinal lesions, several lysosomal cathepsins are known to participate in epithelial permeability in cell-cell connections, such as tight junctions in ulcerative colitis. However, very few studies have focused on the distribution of cathepsins in the esophageal multilayer squamous epithelium. Therefore to clarify the role of cathepsins in RE, we investigated their immunohistological localization in the esophageal epithelium under normal conditions and after RE. Of the cathepsins examined (cathepsins B, C, D, F, H, L, S, and X), granular immunoreactivity for cathepsins B, C, D and L was observed in the control esophageal epithelia; although, their distribution differed depending on the enzyme examined. In the RE model, immunoreactivity of these cathepsins was increased in esophageal epithelial cells and activated macrophages. The immunoreactivity for cathepsins F, H, S and X was barely detectable in the control esophageal epithelium. However, in the RE model, we noticed a slight increase in the expression of cathepsins H and X in the epithelial cells. Furthermore, activated macrophages of the RE model possessed intense immunoreactivity for these cathepsins, which may have been related to esophageal inflammatory mechanisms.

Liver kinase B1 regulates the centrosome via PLK1.

Liver kinase B1 (LKB1) is a tumor suppressor mutationally inactivated in Peutz-Jeghers syndrome (PJS) and various sporadic cancers. Although LKB1 encodes a kinase that possesses multiple functions, no individual hypothesis posed to date has convincingly explained how loss of LKB1 contributes to carcinogenesis. In this report we demonstrated that LKB1 maintains genomic stability through the regulation of centrosome duplication. We found that LKB1 colocalized with centrosomal proteins and was situated in the mitotic spindle pole. LKB1 deficiency-induced centrosome amplification was independent of AMP-activated protein kinase (AMPK), a well-defined substrate of LKB1. Cells lacking LKB1 exhibited an increase in phosphorylated and total Polo-like kinase 1 (PLK-1), NIMA-related kinase 2 (NEK2), and ninein-like protein (NLP). Overexpression of active PLK1 (T210D) reversed the inhibition of LKB1 on centrosome amplification. In contrast, depletion of PLK1 with siRNA or suppression of PLK1 kinase activity with BTO-1 (5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide) abrogated LKB1 deficiency-induced centrosome amplification. We further characterized that LKB1 phosphorylated and activated AMPK-related kinase 5 (NUAK1 or ARK5) that in turn increased the phosphorylation of MYPT1, enhanced the binding between MYPT1-PP1 and PLK1, and conferred an effective dephosphorylation of PLK1. More importantly, we noted that LKB1-deficient cells exhibited multiple nuclear abnormalities, such as mitotic delay, binuclear, polylobed, grape, large, and micronuclear. Immediate depletion of LKB1 resulted in the accumulation of multiploidy cells. Expression of LKB1 is reversely correlated with the levels of PLK1 in human cancer tissues. Thus, we have uncovered a novel function of LKB1 in the maintenance of genomic stability through the regulation of centrosome mediated by PLK1.

Oxidative DNA damage in human esophageal cancer: clinicopathological analysis of 8-hydroxydeoxyguanosine and its repair enzyme.

Both internal and external oxidative stresses act on DNA and can induce carcinogenesis. 8-hydroxydeoxyguanosine (8-OHdG) is an indicator of oxidative stress and it leads to transversion mutations and carcinogenesis. 8-OHdG is excision-repaired by 8-OHdG DNA glycosylase (OGG1). The purpose of this study is to clarify the effect of oxidative DNA damage and repair enzymes on esophageal carcinogenesis. The levels of 8-OHdG and OGG1 were immunohistochemically evaluated in resected specimens, including squamous cell carcinoma (SCC) in 97 patients with esophageal cancer. Higher levels of 8-OHdG in normal esophageal epithelium were associated with a higher smoking index (P = 0.0464). The 8-OHdG level was higher in cancerous areas than in normal epithelia (P = 0.0061), whereas OGG1 expression was weaker in cancerous areas than in normal epithelia (P < 0.0001). An increase of OGG1 expression in normal epithelium was observed as 8-OHdG levels increased (P = 0.0011). However, this correlation was not observed in cancerous areas. High OGG1 expression in the cytoplasm was related to deeper tumors (P = 0.0023), node metastasis (P = 0.0065) and stage (P = 0.0019). Oxidative DNA damage, which is attributable to smoking as well as disturbances in DNA repair systems, appears to be closely related to esophageal carcinogenesis and its progression.

Niche-specific requirement for hyphal wall protein 1 in virulence of Candida albicans.

Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.