Carbonic anhydrases III and IX are new players in the crosstalk between adrenocortical carcinoma and its altered adipose microenvironment.

PURPOSE: Adrenocortical carcinoma (ACC), a rare malignancy of the adrenocortex, is characterized by a crosstalk between the adipose microenvironment and tumor. Here, we assessed the involvement of carbonic anhydrase (CA) enzymes III and IX (CAIII and CAIX), in the metabolic alterations of the adipose tissue characterizing obesity and in the local crosstalk between the tumor adipose microenvironment and ACC. RESULTS/METHODS: CAIII and CAIX expression is altered in visceral adipose tissue (VAT) in obesity and in ACC. A significant CAIX upregulation was present in ACC at advanced stages (n = 14) (fold increase FI = 7.4 ± 0.1, P < 0.05) associated with lower CAIII levels (FI = 0.25 ± 0.06, P < 0.001), compared with lower stages (n = 9). In vitro coculture between visceral adipose stem cells (ASCs) and ACC cell lines, H295R and MUC-1, mimicking the interaction occurring between VAT and advanced ACC, showed a significant CAIX upregulation in H295R but not in MUC-1 cells, and a decreased expression of CAIII. The effect on adipose cells was different when cocultured with H295R or MUC-1 cells. Coculture did not modulate CAIII expression in ASCs, which, however, was significantly downregulated with H295R (FI = 0.34 ± 0.11, P < 0.05) and upregulated by MUC-1 when cocultured ASCs were induced to differentiate toward adipocytes, with an expression profile similar to what found in VAT of obese subjects. CAIX expression was markedly increased in ASCs cocultured with H295R and to a less extent following adipogenesis induction (FI = 150.9 ± 46.5 and FI = 4.6 ± 1.1, P < 0.01, respectively). CONCLUSION: Our findings highlight a modulation of CAIII and CAIX in the metabolic crosstalk between ACC and its local adipose microenvironment, suggesting that CAs might represent a potential target for novel anticancer therapies.

Carbonic anhydrase 3 increases during liver adipogenesis even in pre-obesity, and its inhibitors reduce liver adipose accumulation.

The abnormal lipid metabolism in the liver that occurs after high caloric intake is the main cause of nonalcoholic fatty liver disease (NAFLD). Differences between samples from healthy livers and livers from individuals with NAFLD indicate that changes in liver function occur during disease progression. Here, we examined changes in protein expression in a fatty liver model in the early stages of obesity to identify potential alterations in function. The proteins expressed in the liver tissue of pre-obese rats were separated via SDS/PAGE and stained with Coomassie brilliant blue-G250. Peptide mass fingerprinting indicated an increase in the expression of carbonic anhydrase 3 (CA3) relative to controls. Western blotting analysis confirmed the increase in CA3 expression, even in an early fat-accumulation state in which excessive weight gain had not yet occurred. In human hepatoma HepG2 cells, fat accumulation induced with oleic acid also resulted in increased CA3 expression. When the cells were in a state of fat accumulation, treating them with the CA3 inhibitors acetazolamide (ACTZ) or 6-ethoxyzolamide (ETZ) suppressed fat accumulation, but only ETZ somewhat reduced the fat-induced upregulation of CA3 expression. Expression of CA3 was therefore upregulated in response to the consumption of a high-fat diet, even in the absence of an increase in body weight. The suppression of CA3 activity by ACTZ or ETZ reduced fat accumulation in hepatocytes, suggesting that CA3 is involved in the development of fatty liver.

Carbonic Anhydrase III Attenuates Hypoxia-Induced Apoptosis and Activates PI3K/Akt/mTOR Pathway in H9c2 Cardiomyocyte Cell Line.

Myocardial ischemia can cause insufficient oxygen and functional damage to myocardial cells. Carbonic anhydrase III (CAIII) has been found to be closely related to the abnormality of cardiomyocytes. To investigate the role of CAIII in the apoptosis of myocytes under hypoxic conditions and facilitate the strategy for treating hypoxia-induced damage, in vitro experiments in H9c2 were employed. The protein expression of CAIII in H9c2 cells after hypoxia or normoxia treatment was determined by western blotting and immunohistochemistry. MTT assay was employed for cells viability measurement and LDH release was monitored. The apoptotic cells were observed using immunofluorescence assay, flow cytometric analysis, and TUNEL assay. CAIII-overexpression or -knockdown cells were constructed to determine the role of CAIII in regulating apoptosis-related proteins caspase-3, Bax, Bcl-2, and anti-apoptosis pathway PI3K/Akt/mTOR. The mRNA levels of CAIII and genes related to CAIII synthesis including REN, IGHM, APOBEC 3F, and SKOR2 were significantly upregulated in hypoxia fetal sheep. The expression of CAIII protein and content of apoptotic H9c2 cells were increased at 1, 3, 6, and 12 h after hypoxia treatment. Overexpression of CAIII significantly upregulated Bcl2 level and downregulated Bax and caspase-3 cleavage levels, while its knockdown led to the contrary results. Overexpressed CAIII promoted the HIF-1α level and activated the PI3K/Akt/mTOR pathway, thereby exerting an inhibitory effect on hypoxia-induced apoptosis. In conclusion, our findings revealed that CAIII could protect cell from hypoxia-apoptosis of H9c2 cells, in which, activated PI3K/Akt/mTOR signaling pathway may be involved.

Carbonic anhydrase III is a new target of HIF1α in prostate cancer model.

Carbonic Anhydrase III (CAIII) belongs to a member of the alpha Carbonic Anhydrase (CA) family. Although some CA members are strongly up-regulated by HIF1-α, it is not known about the transcriptional regulation of CAIII in prostate cancer cells, PCa. Therefore, we aimed to identify regulatory regions important for the regulation of CAIII gene under hypoxic conditions in human prostate cancer cells (PC3). The present study, for the first time, demonstrated that the chemically mimicked hypoxic condition led to the induced CAIII mRNA and protein expression in prostate cancer cells. Transcriptional regulation of CAIII was investigated by transient transfection assay that indicates that the most active promoter activity was in the region of P2 -699/+86. Hypoxic condition also upregulates the basal activity of for P1;-941/+86 and P2;-699/+86 constructs containing putative Hypoxia Response Element (HRE) region located in -268/-252. EMSA analysis of HRE located in -268/-252 bases, showed one DNA-protein binding complexes. Competition assays indicated this complex is resulted from HIF1α interactions. In addition, site-directed mutagenesis of potential HIF1α binding sites diminished a DNA-protein complex. These findings suggest that CAIII is a hypoxia-regulated gene and valuable for targeting of prostate cancer tumors in hypoxic condition.

Carbonic Anhydrase III Promotes Cell Migration and Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma.

Epithelial-mesenchymal transition (EMT) is strongly correlated with tumor metastasis and contains several protein markers, such as E-cadherin. Carbonic anhydrase III (CA III) exhibits low carbon dioxide hydratase activity in cancer. However, the detailed mechanisms of CA III and their roles in oral cancer are still unknown. This study established a CA III-overexpressed stable clone and observed the expression of CA III protein in human SCC-9 and SAS oral cancer cell lines. The migration and invasion abilities were determined using a Boyden chamber assay. Our results showed that the overexpression of CA III protein significantly increased the migration and invasion abilities in oral cancer cells. Moreover, a whole genome array analysis revealed that CA III regulated epithelial-mesenchymal transition by reducing the expression of epithelial markers. Data from the GEO database also demonstrated that CA III mRNA is negatively correlated with CDH1 mRNA. Mechanistically, CA III increased the cell motility of oral cancer cells through the FAK/Src signaling pathway. In conclusion, this suggests that CA III promotes EMT and cell migration and is potentially related to the FAK/Src signaling pathway in oral cancer.

Structural Mapping of Anion Inhibitors to ß-Carbonic Anhydrase psCA3 from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative facultative anaerobe belonging to the Pseudomonadaceae family. It is a multidrug-resistant opportunistic human pathogen, a common cause of life-threatening nosocomial infections, and a key bacterial agent in cystic fibrosis and endocarditis. The bacterium exhibits intrinsic resistance to most antibacterial agents, including aminoglycosides and quinolones. Hence, the identification of new drug targets for P. aeruginosa is ongoing. PsCA3 is a ß-class carbonic anhydrase (ß-CA) that catalyzes the reversible hydration of carbon dioxide to bicarbonate and represents a new class of antimicrobial target. Previously, inhibitor screening studies of psCA3 have shown that a series of small anions including sulfamide (SFN), imidazole (IMD), and 4-methylimidazole (4MI), and thiocyanate (SCN) inhibit the enzyme with efficiencies in the micro- to millimolar range. Herein the X-ray crystal structures of these inhibitors in complex with psCA3 are presented and compared with human CA II. This structural survey into the binding modes of small anions forms the foundation for the development of inhibitors against ß-CAs and more selective inhibitors against P. aeruginosa.

Protective Role of Carbonic Anhydrases III and VII in Cellular Defense Mechanisms upon Redox Unbalance.

Under oxidative stress conditions, several constitutive cellular defense systems are activated, which involve both enzymatic systems and molecules with antioxidant properties such as glutathione and vitamins. In addition, proteins containing reactive sulfhydryl groups may eventually undergo reversible redox modifications whose products act as protective shields able to avoid further permanent molecular oxidative damage either in stressful conditions or under pathological circumstances. After the recovery of normal redox conditions, the reduced state of protein sulfhydryl groups is restored. In this context, carbonic anhydrases (CAs) III and VII, which are human metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate and proton, have been identified to play an antioxidant role in cells where oxidative damage occurs. Both proteins are mainly localized in tissues characterized by a high rate of oxygen consumption, and contain on their molecular surface two reactive cysteine residues eventually undergoing S-glutathionylation. Here, we will provide an overview on the molecular and functional features of these proteins highlighting their implications into molecular processes occurring during oxidative stress conditions.

Glutathiolation Triggers Proteins for Degradation by the Ubiquitin- Proteasome Pathway.

BACKGROUND: Glutathione is a small antioxidant peptide in cells and it plays an important role in maintaining a reducing intracellular environment. Glutathione is also involved in the dynamic regulation of specific protein functions by reversible glutathiolation of certain proteins in response to oxidative stress. OBJECTIVE: The purpose of this work is to mechanistically investigate the effects of glutathiolation on the susceptibility of proteins to degradation by the ubiquitinproteasome pathway (UPP). METHODS AND RESULTS: The data show that γC-crystallin and carbonic anhydrase III were barely degraded by the UPP without modifications, but both were rapidly degraded by the UPP after glutathiolation. Modifications of sulfhydryls by other thiol-modification reagents, such as iodoacetamide, also increased the degradation of γC-crystallin, but not as effectively as glutathiolation. Biophysical analysis showed that glutathiolation caused reversible conformational changes of these proteins, including a significant increase in protein surface hydrophobicity and a decrease in thermal stability. The modified protein regained its native conformation and its resistance to degradation upon removal of the glutathione moiety. A cataract-causing T5P mutant γC-crystallin shares many biophysical characteristics as glutathiolated γC-crystallin, including increased surface hydrophobicity and decreased thermal stability. T5P mutant γC-crystallin was also rapidly degraded. Comparison of the conformational changes and the susceptibility to degradation of glutathiolated γC-crystallin with other forms of modified γC-crystallin suggests that the glutathiolation-induced exposure of hydrophobic patches, rather than the modification per se, serves as the signal for degradation by the UPP. Consistent with this hypothesis, masking the surface hydrophobicity of glutathiolated and T5P mutant γC-crystallins significantly reduced their susceptibility to degradation by the UPP. CONCLUSION: This work demonstrates that glutathiolation is a novel mechanism for the UPP to recognize substrates in response to oxidative stress.

Expression of carbonic anhydrase III and skeletal muscle remodeling following selective denervation.

Carbonic anhydrase III (CAIII) is expressed selectively in type I (slow­twitch) myofibers. To investigate the association between changes in the expression of CAIII and skeletal muscle structure following denervation, the present study stained adjacent sections of skeletal muscle for ATPase and immunohistochemically for CAIII. In addition, differences in the protein expression and phosphatase activity of CAIII were examined by western blot and phosphatase staining between rat soleus and extensol digitorum longus (EDL) muscles, which are composed of predominantly slow­ and fast­twitch fibers, respectively. Upon denervation, the EDL muscle showed more pronounced structural changes, compared with the soleus muscle. There was a transformation from fast to slow fibers, and a concomitant increase in fibers positive for CAIII. Following denervation, the protein expression of CAIII initially increased and then decreased in the soleus muscle, whereas the protein expression of CAIII in the EDL muscle increased gradually with time. In contrast to the protein changes, phosphatase activity in the soleus and EDL muscles decreased significantly following denervation. These results indicated that, following denervation, changes in the expression of CAIII were associated with myofiber remodeling. Specifically, the change in the expression of CAIII reflected the conversion to type I myofibers, suggesting the importance of CAIII in resistance to fatigue in skeletal muscle.

Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis.

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.

Effects of pepsin A on heat shock protein 70 response in laryngopharyngeal reflux patients with chronic rhinosinusitis.

OBJECTIVES: We investigated the relationship between laryngopharyngeal reflux (LPR) and chronic rhinosinusitis (CRS), and explored the effects of pepsin A on the level of heat shock protein 70 (HSP70) in CRS. METHODS: We included 23 CRS patients with nasal polyps (CRSwNP), 26 CRS patients without nasal polyps (CRSsNP) and nine normal controls to measure pepsin A levels in nasal secretions, blood plasma and nasal tissues, to measure HSP70 levels in nasal tissues, and to detect pepsinogen A, HSPA5, cyclo-oxygenase-2 (COX-2), and carbonic anhydrase III (CAIII) mRNA expression levels in nasal tissues. RESULTS: Pepsin A levels in nasal secretions were significantly higher in CRSwNP/CRSsNP patients than in controls. HSP70 levels were significantly increased in pepsin A-positive turbinate mucosa compared to controls (p < .001). Similarly, HSP70 levels were significantly increased in pepsin A-positive polyp tissues than in pepsin A-negative polyp tissues (p = .016). Furthermore, no association was found between the presence of pepsin A and HSPA5, COX-2, and CAIII mRNA expression levels. CONCLUSIONS: These results suggest that LPR may play a role in the development of CRS through pepsin A reflux, and increased HSP70 expression may be associated with the pathogenic mechanism of mucosal injury in CRS.

Identification of consensus biomarkers for predicting non-genotoxic hepatocarcinogens.

The assessment of non-genotoxic hepatocarcinogens (NGHCs) is currently relying on two-year rodent bioassays. Toxicogenomics biomarkers provide a potential alternative method for the prioritization of NGHCs that could be useful for risk assessment. However, previous studies using inconsistently classified chemicals as the training set and a single microarray dataset concluded no consensus biomarkers. In this study, 4 consensus biomarkers of A2m, Ca3, Cxcl1, and Cyp8b1 were identified from four large-scale microarray datasets of the one-day single maximum tolerated dose and a large set of chemicals without inconsistent classifications. Machine learning techniques were subsequently applied to develop prediction models for NGHCs. The final bagging decision tree models were constructed with an average AUC performance of 0.803 for an independent test. A set of 16 chemicals with controversial classifications were reclassified according to the consensus biomarkers. The developed prediction models and identified consensus biomarkers are expected to be potential alternative methods for prioritization of NGHCs for further experimental validation.

Expression of CAIII and Hsp70 Is Increased the Mucous Membrane of the Posterior Commissure in Laryngopharyngeal Reflux Disease.

PURPOSE: We tried to evaluate the difference in the expression of carbonic anhydrase (CA) III and heat shock protein (Hsp) 70 between laryngopharyngeal reflux disease (LPRD) and non-LPRD patients. MATERIALS AND METHODS: The study involved 28 patients who underwent laryngeal microsurgery due to benign laryngeal disease from March to August 2008. Reflux symptom index (RSI) and reflux finding score (RFS) were measured for each person, and they were assigned either to the LPRD group (n=10) or non-LPRD group (n=18). Tissue samples were obtained from the mucosa of posterior commissure, and immunohistochemistry (IHC) staining of CAIII and Hsp70 was performed. The IHC scores were measured and compared with clinical features including RSI and RFS. RESULTS: Total 10 patients were assigned as LPRD group, and 18 patients were as control group. The mean IHC score of CAIII and Hsp70 was 1.70 ± 1.06 and 1.90 ± 0.88, respectively, in LPRD patients, whereas the mean IHC score of CAIII and Hsp70 was 0.78 ± 0.73 and 0.94 ± 0.87, respectively, in non-LPRD patients. The difference between two groups was statistically significant (p<0.05). CONCLUSION: CAIII and Hsp70 expressions were higher in LPRD patients that in non-LPRD patients, suggesting the possibility as one of biomomarker in LPRD diagnosis.

In-depth phenotyping of a Donnai-Barrow patient helps clarify proximal tubule dysfunction.

BACKGROUND: The megalin/cubilin/amnionless complex is essential for albumin and low molecular weight (LMW) protein reabsorption by renal proximal tubules (PT). Mutations of the LRP2 gene encoding megalin cause autosomal recessive Donnai-Barrow/facio-oculo-acoustico-renal syndrome (DB/FOAR), which is characterized by LMW proteinuria. The pathophysiology of DB/FOAR-associated PT dysfunction remains unclear. CLINICAL CASE: A 3-year-old girl presented with growth retardation and proteinuria. Clinical examination was unremarkable, except for a still-opened anterior fontanel and myopia. Psychomotor development was delayed. At 6, she developed sensorineural hearing loss. Hypertelorism was noted when she turned 12. Blood analyses, including renal function parameters, were normal. Urine sediment was bland. Proteinuria was significant and included albumin and LMW proteins. Immunoblotting analyses detected cubilin and type 3 carbonic anhydrase (CA3) in the urine. Renal ultrasound was unremarkable. Optical examination of a renal biopsy did not disclose any tubular or glomerular abnormality. Electron microscopy revealed that PT apical endocytic apparatus was significantly less developed. Immunostaining for megalin showed a faint signal in PT cytosol contrasting with the distribution of cubilin at the apical membrane. The diagnostic procedure led to identifying two mutations of the LRP2 gene. CONCLUSIONS: The functional loss of megalin in DB/FOAR causes PT dysfunction characterized by increased urinary shedding of CA3 and cubilin.

Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and (3)H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼ 75-80 kDa, ∼ 95-100 kDa, and ∼ 155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼ 160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼ 2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

Sox10 expressing cells in the lateral wall of the aged mouse and human cochlea.

Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1-3 month-old) and aged (2-2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10(+) cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10(+) marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10(+) cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10(+) cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10(+) cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear.

Anti-carbonic anhydrase III autoantibodies in vasculitis syndrome.

AIM: To identify autoantibodies useful in the diagnosis of primary vasculitides. METHODS: The presence of antibodies against proteins in the lysate of mouse blood vessels was examined by two-dimensional electrophoresis followed by Western blotting for the pooled serum sample from patients with various forms of vasculitis: polyarteritis nodosa (PAN), microscopic polyangiitis (MPA), Wegener's granulomatosis (WG) and Takayasu's arteritis (TA). Autoantigenicity in patients with vasculitides was examined by Western blotting and enzyme-linked immunosorbent assay (ELISA). Clinicopathological correlations between the positivity of the autoantibodies and clinical status of patients with the vasculitis were examined. RESULTS: The autoantigen detected in the lysate of pooled sera from patients with vasculitides was identified by mass spectrometry as carbonic anhydrase III (CAIII). ELISA showed significantly higher prevalence of anti-CAIII antibodies in MPA patients (MPA, 11/23 [47.8%]; healthy controls, 2/32 [6.3%]; P < 0.001). Further, anti-CAIII antibody-positive MPA patients had higher vasculitis activity scores compared to anti-CAIII antibody-negative patients, and a weak and not significant negative correlation was observed between anti-CAIII antibody levels and myeloperoxidase - anti-nuclear cytoplasmic antibody (MPO-ANCA) levels. No significant differences were found in anti-CAIII autoantibody levels between MPA and the other primary vasculitides. CONCLUSION: We found significantly high prevalence of anti-CAIII antibody levels in sera from MPA patients. Although the number of samples available in this study is small and anti-CAIII autoantibodies display weak specificity for MPA, anti-CAIII antibodies may be useful for diagnosing MPA in patients who have no ANCA, as well as for assessing disease activity.

Carbonic anhydrases III and IV autoantibodies in rheumatoid arthritis, systemic lupus erythematosus, diabetes, hypertensive renal disease, and heart failure.

In the present study, the CA III and IV autoantibodies, CA activity, antioxidant enzymes and cytokines in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), diabetes, hypertensive renal disease, and heart failure were investigated. The anti-CA III antibody titers in patients with RA, SLE, and type 1 diabetes (T1D) were significantly higher than that in control groups (P < 0.05). The anti-CA IV antibody titers in patients with RA, SLE, type 1 diabetic nephropathy (T1DN), and heart failure were significantly higher than that in control groups (P < 0.05) while anti-CA IV antibody could suppress the total CA activity. The SOD and GPx levels in patients with RA, SLE, and T1DN were significantly lower than that in control groups (P < 0.05). IL-6, IL-17, IFN-γ, and TNF-α levels were significantly higher in SLE group compared with the control group (P < 0.05). Weak but significant correlations were found between anti-CA III antibodies and ESR in RA (r = 0.403, P = 0.013) and SLE patients (r = 0.397, P = 0.007). These results suggested that the generation of CA III and IV autoantibodies, antioxidant enzymes, and cytokines might influence each other and CA autoantibodies might affect the normal physiology function of CA.

Molecular characterization of EGFR and EGFRvIII signaling networks in human glioblastoma tumor xenografts.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ≈ 50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2-7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.

Biomarkers and laryngopharyngeal reflux.

Laryngopharyngeal reflux is a controversial but increasingly made diagnosis used in patients with a collection of often non-specific laryngeal symptoms. It is a clinical diagnosis, and its pathophysiology is currently poorly understood. Previous reflux research has focused on injurious agents, acid, pepsin and biomarker expression. Failure of intrinsic defences in the larynx may cause changes in laryngeal epithelia, particularly alterations in carbonic anhydrases and E-cadherin. Carbonic anhydrase III levels vary in the larynx in response to laryngopharyngeal reflux, depending on location. Expression of E-cadherin, a known tumour suppressor, is reduced in the presence of reflux. Mucin expression also varies according to the severity of reflux. Further research is required to define the clinical entity of laryngopharyngeal reflux, and to identify a definitive mechanism for mucosal injury. Understanding this mechanism should allow the development of a comprehensive model, which would enable future diagnostic and therapeutic interventions to be developed.