S-9-PAHSA's neuroprotective effect mediated by CAIII suppresses apoptosis and oxidative stress in a mouse model of type 2 diabetes.

BACKGROUND: With the rapidly increasing prevalence of metabolic diseases such as type 2 diabetes mellitus (T2DM), neuronal complications associated with these diseases have resulted in significant burdens on healthcare systems. Meanwhile, effective therapies have remained insufficient. A novel fatty acid called S-9-PAHSA has been reported to provide metabolic benefits in T2DM by regulating glucose metabolism. However, whether S-9-PAHSA has a neuroprotective effect in mouse models of T2DM remains unclear. METHODS: This in vivo study in mice fed a high-fat diet (HFD) for 5 months used fasting blood glucose, glucose tolerance, and insulin tolerance tests to examine the effect of S-9-PAHSA on glucose metabolism. The Morris water maze test was also used to assess the impact of S-9-PAHSA on cognition in the mice, while the neuroprotective effect of S-9-PAHSA was evaluated by measuring the expression of proteins related to apoptosis and oxidative stress. In addition, an in vitro study in PC12 cells assessed apoptosis, oxidative stress, and mitochondrial membrane potential with or without CAIII knockdown to determine the role of CAIII in the neuroprotective effect of S-9-PAHSA. RESULTS: S-9-PAHSA reduced fasting blood glucose levels significantly, increased insulin sensitivity in the HFD mice and also suppressed apoptosis and oxidative stress in the cortex of the mice and PC12 cells in a diabetic setting. By suppressing oxidative stress and apoptosis, S-9-PAHSA protected both neuronal cells and microvascular endothelial cells in in vivo and in vitro diabetic environments. Interestingly, this protective effect of S-9-PAHSA was reduced significantly when CAIII was knocked down in the PC12 cells, suggesting that CAIII has a major role in the neuroprotective effect of S-9-PAHSA. However, overexpression of CAIII did not significantly enhance the protective effect of S-9-PAHSA. CONCLUSION: S-9-PAHSA mediated by CAIII has the potential to exert a neuroprotective effect by suppressing apoptosis and oxidative stress in neuronal cells exposed to diabetic conditions. Furthermore, S-9-PAHSA has the capability to reduce fasting blood glucose and LDL levels and enhance insulin sensitivity in mice fed with HFD.

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.

Tip60 might be a candidate for the acetylation of hepatic carbonic anhydrase I and III in mice.

BACKGROUND: Carbonic anhydrases (CAs) play a significant role in maintaining pH balance by catalyzing the conversion of carbon dioxide to bicarbonate. The regulation of pH is critical for all living organisms. Although there are many studies in the literature on the biochemical, functional, and structural features of CAs, there is not sufficient information about the epigenetic regulation of CAs. METHODS AND RESULTS: The lysine acetyltransferase TIP60 (60 kDa Tat-interactive protein) was knocked out specifically in mouse liver using the Cre/loxP system, and knockout rate was shown as 83-88% by Southern blot analysis. The impact of Tip60 on the expression of Ca1, Ca3, and Ca7 was investigated at six Zeitgeber time (ZT) points in the control and liver-specific Tip60 knockout mice (mutant) groups by real-time PCR. In the control group, while Ca1 showed the highest expression at ZT8 and ZT12, the lowest expression profile was observed at ZT0 and ZT20. Hepatic Ca1 displayed robust circadian expression. However, hepatic Ca3 exhibited almost the same level of expression at all ZT points. The highest expression of Ca7 was observed at ZT12, and the lowest expression was determined at ZT4. Furthermore, hepatic Ca7 also showed robust circadian expression. The expression of Ca1 and Ca3 significantly decreased in mutant mice at all time periods, but the expression of Ca7 used as a negative control was not affected. CONCLUSIONS: It was suggested for the first time that Tip60 might be considered a candidate protein in the regulation of the Ca1 and Ca3 genes, possibly by acetylation.

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.

Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

BACKGROUNDS & AIMS: Squalene epoxidase (SQLE) is the rate-limiting enzyme for cholesterol biosynthesis. We elucidated the functional significance, molecular mechanisms, and clinical impact of SQLE in nonalcoholic steatohepatitis (NASH). METHODS: We performed studies with hepatocyte-specific Sqle overexpression transgenic (Sqle tg) mice and mice given high-fat high-cholesterol (HFHC) or methionine- and choline-deficient (MCD) diet to induce NASH. SQLE downstream target carbonic anhydrase III (CA3) was identified using co-immunoprecipitation and Western Blot. Some mice were given SQLE inhibitor (terbinafine) and CA3 inhibitor (acetazolamide) to study the therapeutic effects in NASH. Human samples (N = 217) including 65 steatoses, 80 NASH, and 72 healthy controls were analyzed for SQLE levels in liver tissue and in serum. RESULTS: SQLE is highly up-regulated in human NASH and mouse models of NASH. Sqle tg mice triggered spontaneous insulin resistance, hepatic steatosis, liver injury, and accelerated HFHC or MCD diet-induced NASH development. Mechanistically, SQLE tg mice caused hepatic cholesterol accumulation, thereby triggering proinflammatory nuclear factor-κB signaling and steatohepatitis. SQLE directly bound to CA3, which induced sterol regulatory element-binding protein 1C activation, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase1 expression and de novo hepatic lipogenesis. Combined targeting SQLE (terbinafine) and CA3 (acetazolamide) synergistically ameliorated NASH in mice with superior efficacy to either drug alone. Serum SQLE with CA3 could distinguish patients with NASH from steatosis and healthy controls (area under the receiver operating characteristic curve, 0.815; 95% confidence interval, 0.758-0.871). CONCLUSIONS: SQLE drives the initiation and progression of NASH through inducing cholesterol biosynthesis, and SQLE/CA3 axis-mediated lipogenesis. Combined targeting of SQLE and CA3 confers therapeutic benefit in NASH. Serum SQLE and CA3 are novel biomarkers for the noninvasive diagnosis of patients with NASH.

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.

Synthesis and evaluation of 2,4,5-trisubstitutedthiazoles as carbonic anhydrase-III inhibitors.

A series of 17 compounds (12-16 b) with 2,4,5-trisubstitutedthiazole scaffold having 5-aryl group, 4-carboxylic acid/ester moiety, and 2-amino/amido/ureido functional groups were synthesised, characterised, and evaluated for their carbonic anhydrase (CA)-III inhibitory activities using the size exclusion Hummel-Dreyer method (HDM) of chromatography. Compound 12a with a free amino group at the 2-position, carboxylic acid moiety at the 4-position, and a phenyl ring at the 5-position of the scaffold was found to be the most potent CA-III inhibitor (Ki = 0.5 µM). The presence of a carboxylic acid group at the 4-position of the scaffold was found to be crucial for the CA-III inhibitory activity. Furthermore, replacement of the free amino group with an amide and urea group resulted in a significant reduction of activity (compounds 13c and 14c, Ki = 174.1 and 186.2 µM, respectively). Thus, compound 12a (2-amino-5-phenylthiazole-4-carboxylic acid) can be considered as the lead molecule for further modification and development of more potent CA-III inhibitors.

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.

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.

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.

Expression of Carbonic Anhydrase III, a Nucleus Pulposus Phenotypic Marker, is Hypoxia-responsive and Confers Protection from Oxidative Stress-induced Cell Death.

The integrity of the avascular nucleus pulposus (NP) phenotype plays a crucial role in the maintenance of intervertebral disc health. While advances have been made to define the molecular phenotype of healthy NP cells, the functional relevance of several of these markers remains unknown. In this study, we test the hypothesis that expression of Carbonic Anhydrase III (CAIII), a marker of the notochordal NP, is hypoxia-responsive and functions as a potent antioxidant without a significant contribution to pH homeostasis. NP, but not annulus fibrosus or end-plate cells, robustly expressed CAIII protein in skeletally mature animals. Although CAIII expression was hypoxia-inducible, we did not observe binding of HIF-1α to select hypoxia-responsive-elements on Car3 promoter using genomic chromatin-immunoprecipitation. Similarly, analysis of discs from NP-specific HIF-1α null mice suggested that CAIII expression was independent of HIF-1α. Noteworthy, silencing CAIII in NP cells had no effect on extracellular acidification rate, CO2 oxidation rate, or intracellular pH, but rather sensitized cells to oxidative stress-induced death mediated through caspase-3. Our data clearly suggests that CAIII serves as an important antioxidant critical in protecting NP cells against oxidative stress-induced injury.

Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.

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.

Bioactive Natural Product and Superacid Chemistry for Lead Compound Identification: A Case Study of Selective hCA III and L-Type Ca2+ Current Inhibitors for Hypotensive Agent Discovery.

Dodoneine (Ddn) is one of the active compounds identified from Agelanthusdodoneifolius, which is a medicinal plant used in African pharmacopeia and traditional medicine for the treatment of hypertension. In the context of a scientific program aiming at discovering new hypotensive agents through the original combination of natural product discovery and superacid chemistry diversification, and after evidencing dodoneine's vasorelaxant effect on rat aorta, superacid modifications allowed us to generate original analogues which showed selective human carbonic anhydrase III (hCA III) and L-type Ca2+ current inhibition. These derivatives can now be considered as new lead compounds for vasorelaxant therapeutics targeting these two proteins.

Carbonic anhydrase III (Car3) is not required for fatty acid synthesis and does not protect against high-fat diet induced obesity in mice.

Carbonic anhydrases are a family of enzymes that catalyze the reversible condensation of water and carbon dioxide to carbonic acid, which spontaneously dissociates to bicarbonate. Carbonic anhydrase III (Car3) is nutritionally regulated at both the mRNA and protein level. It is highly enriched in tissues that synthesize and/or store fat: liver, white adipose tissue, brown adipose tissue, and skeletal muscle. Previous characterization of Car3 knockout mice focused on mice fed standard diets, not high-fat diets that significantly alter the tissues that highly express Car3. We observed lower protein levels of Car3 in high-fat diet fed mice treated with niclosamide, a drug published to improve fatty liver symptoms in mice. However, it is unknown if Car3 is simply a biomarker reflecting lipid accumulation or whether it has a functional role in regulating lipid metabolism. We focused our in vitro studies toward metabolic pathways that require bicarbonate. To further determine the role of Car3 in metabolism, we measured de novo fatty acid synthesis with in vitro radiolabeled experiments and examined metabolic biomarkers in Car3 knockout and wild type mice fed high-fat diet. Specifically, we analyzed body weight, body composition, metabolic rate, insulin resistance, serum and tissue triglycerides. Our results indicate that Car3 is not required for de novo lipogenesis, and Car3 knockout mice fed high-fat diet do not have significant differences in responses to various diets to wild type mice.

Cryoannealing-induced space-group transition of crystals of the carbonic anhydrase psCA3.

Cryoannealing has been demonstrated to improve the diffraction quality and resolution of crystals of the ß-carbonic anhydrase psCA3 concomitant with a change in space group. After initial flash-cooling in a liquid-nitrogen cryostream an X-ray diffraction data set from a psCA3 crystal was indexed in space group P21212 and was scaled to 2.6 Šresolution, but subsequent cryoannealing studies revealed induced protein rearrangements in the crystal contacts, which transformed the space group to I222, with a corresponding improvement of 0.7 Šin resolution. Although the change in diffraction resolution was significant, only minor changes in the psCA3 structure, which retained its catalytic `open' conformation, were observed. These findings demonstrate that cryoannealing can be successfully utilized to induce higher diffraction-quality crystals while maintaining enzymatically relevant conformations and may be useful as an experimental tool for structural studies of other enzymes where the initial diffraction quality is poor.

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.