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Background: Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2) to bicarbonate and a proton, and alpha-class CA has been reported to facilitate the acid acclimation of Helicobacter pylori (hpalphaCA). The purpose of this study was to characterize the beta-class CA of H. pylori (hpbetaCA) and elucidate the role of this enzyme as a possible drug target for eradication therapy.Methods: We isolated DNA clones of independent H. pylori strains obtained from patients with gastritis (n = 15), gastric ulcer (n = 6), or gastric cancer (n = 16), and then studied genetic polymorphisms. In addition, the susceptibility of H. pylori to sulpiride, an antiulcer drug and efficient inhibitor of both hpalphaCA and hpbetaCA, was studied with an in vitro killing assay.Results: DNA sequences of all 37 hpbetaCA clones encoded a 221 amino acid polypeptide with a variety of polymorphisms (57 types of amino acid substitution at 48 residue positions). There was no polymorphism functionally relevant to the gastric lesion type. One strain included unique residues that were not seen in the other 36 clones from Japanese patients but which were found in a strain obtained from the United Kingdom. Sulpiride had killing effects at concentrations greater than 200 microg/ml for H. pylori, including strains resistant to clarithromycin, metronidazole, or ampicillin.Conclusions: Helicobacter pylori might have evolved independently in the Caucasian and Japanese populations. Dual inhibition of alpha-and beta-class CAs could be applied as alternative therapy for eradication of H. pylori. [ABSTRACT FROM AUTHOR]

Social behaviors often consist of a motivational phase followed by action. Here we show that neurons in the ventromedial hypothalamus ventrolateral area (VMHvl) of mice encode the temporal sequence of aggressive motivation to action. The VMHvl receives local inhibitory input (VMHvl shell) and long-range input from the medial preoptic area (MPO) with functional coupling to neurons with specific temporal profiles. Encoding models reveal that during aggression, VMHvl shell vgat+ activity peaks at the start of an attack, whereas activity from the MPO-VMHvl vgat+ input peaks at specific interaction endpoints. Activation of the MPO-VMHvl vgat+ input promotes and prolongs a low motivation state, whereas activation of VMHvl shell vgat+ results in action-related deficits, acutely terminating attack. Moreover, stimulation of MPO-VMHvl vgat+ input is positively valenced and anxiolytic. Together, these data demonstrate how distinct inhibitory inputs to the hypothalamus can independently gate the motivational and action phases of aggression through a single locus of control., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Hand-gripping training is important for improving the fundamental functions of human physical activity. Bernstein's idea of "repetition without repetition" suggests that motor control function should be trained under changing states. The randomness level of load should be visualized for self-administered screening when repeating various training tasks under changing states. This study aims to develop a sensing methodology of random loads applied to both the agonist and antagonist skeletal muscles when performing physical tasks. We assumed that the time-variability and periodicity of the applied load appear in the time-series feature of muscle deformation data. In the experiment, 14 participants conducted the gripping tasks with a gripper, ball, balloon, Palm clenching, and paper. Crumpling pieces of paper (paper exercise) involves randomness because the resistance force of the paper changes depending on the shape and layers of the paper. Optical myography during gripping tasks was measured, and time-series features were analyzed. As a result, our system could detect the random movement of muscles during training.

In mid-September 2019, a teenage Chinese male student and part-time waiter in Tokyo was diagnosed with multidrug-resistant (MDR) sputum smear-positive pulmonary tuberculosis (TB). This study describes the outbreak investigation of his friends and colleagues at the restaurant. We investigated 6 friends and 15 colleagues; 5 friends and 13 colleagues underwent interferon-γ release assay (IGRA). Of these, 3 friends (60.0%) and 4 colleagues (30.8%) were IGRA-positive. Each of the friends and colleagues was found to have MDR-TB (20% and 7.7%, respectively). Challenges during the investigation were the unavailability of regimens for latent TB infection (LTBI) for contacts with MDR-TB, budgetary constraints concerning implementing computed tomography (CT) scans for the contacts, frequent address changes of foreign-born patients and contacts, investigation during the coronavirus disease pandemic, and variations of alphabetical expression of the names of the patients and contacts, particularly for those from China. It is recommended that the national government officially adopt prophylaxis regimens for LTBI with MDR-TB, address the budgetary constraints regarding CT scans, and deploy liaison officers for coordinating investigations involving many foreign-born patients and contacts scattered in multiple municipalities. The names of foreign-born persons could more accurately be identified using both the alphabet and Chinese characters.

Background: Functional magnetic resonance imaging (fMRI) in mice is typically performed under anesthesia due to difficulties in holding the head of awake mice stably with a conventional three-point fixation method that uses a tooth-bar and earplugs. Although some studies have succeeded in fMRI in awake mice by attaching a head-post on the skull, this cannot be applied to fMRI using a high signal-to-noise ratio (SNR) cryogenic MRI-detector, CryoProbe, because it covers the head of a mouse closely., New Method: We developed head-fixation implements for awake mice that are applicable to fMRI using CryoProbe., Results: A head-bar was surgically attached to the skull of a mouse that was then habituated to a mock fMRI-environment, two hours/day for eight days with physiological examinations of body-weight, fecal weight, electromyogram (EMG), and electrocardiogram. EMG power decreased with just one day of habituation, whereas heart rate decreased after at least seven days of habituation. Estimated head motions of awake mice during fMRI were significantly smaller than a voxel size. Unexpectedly, temporal SNR of fMRI signals for awake mice was higher than that for anesthetized mice held by a conventional method. Functional connectivity in the brain of both anesthetized and awake mice showed bilateral and unilateral networks. COMPARISON WITH EXISTING METHOD(S): fMRI using CryoProbe had been performed on anesthetized mice previously. Our method does not use anesthetics during habituation or fMRI., Conclusion: Our method would be beneficial for translational research using fMRI in mice and humans because human fMRI is typically performed without anesthetics., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Two β-carbonic anhydrases (CAs, EC 4.2.1.1) were identified, cloned and purified in the pathogenic bacterium Legionella pneumophila, denominated LpCA1 and LpCA2. They efficiently catalyze CO2 hydration to bicarbonate and protons, with kcat in the range of (3.4-8.3) × 10(5)s(-1) and kcat/Km of (4.7-8.5) × 10(7)M(-1)s(-1), and are inhibited by sulfonamides and sulfamates. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide(KIs in the range of 40.3-90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide and dichlorophenamide (KIs in the range of 25.2-88.5 nM). As these enzymes may be involved in pH regulation in the phagosome during Legionella infection, their inhibition may lead to antibacterials with a novel mechanism of action., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We investigated the cloning, catalytic activity and anion inhibition of the β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Legionella pneumophila. Two such enzymes, lpCA1 and lpCA2, were found in the genome of this pathogen. These enzymes were determined to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4-8.3)×10(5) s(-1) and kcat/KM values of (4.7-8.5)×10(7) M(-1) s(-1). A set of inorganic anions and small molecules was investigated to identify inhibitors of these enzymes. Perchlorate and tetrafluoroborate were not acting as inhibitors (KI >200 mM), whereas sulfate was a very weak inhibitor for both lpCA1 and lpCA2 (KI values of 77.9-96.5 mM). The most potent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide, diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KI values ranging from 6 to 94 μM. The most potent lpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with KI values ranging from 2 to 13 μM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial agents with a novel mechanism of action., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the gene product, ataxin-3. Microarray analysis revealed a dramatic differential expression of carbonic anhydrase-related protein XI (CA-RPXI/CA11) in the presence or absence of mutant ataxin-3. Therefore, we examined the expression and distribution of all three CA-RPs (CA8, 10, and 11) in human neuronal cells that stably express mutant ataxin-3. Compared with the cells containing normal ataxin-3, protein expression of CA8 and CA11 is significantly increased in human neuroblastoma cells harboring mutant ataxin-3. Semi-quantitative RT-PCR demonstrated that all three CA-RPs exhibited significantly higher transcript levels in neuronal cells expressing mutant ataxin-3. Interestingly, CA11 is distributed not only in the cytoplasm but also within the nuclei of the stably transfected mutant cells when compared with the sole cytoplasmic distribution in cells containing normal ataxin-3. In addition, results from transient transfection assays in SK-N-SH and Neuro2a (N2a) cells also confirmed the nuclear localization of CA11 in the presence of truncated ataxin-3. Most importantly, immunohistochemical staining of the MJD transgenic mouse and post-mortem MJD human brain also revealed that CA11 is highly expressed in both cytoplasm and nuclei of the brain cells. Recruitment of CA11 into nuclear inclusions containing mutant ataxin-3 revealed a possible correlation between CA11 and disease progression. Although the exact function of CA-RPs is still undefined in the central nervous system, our findings suggest that CA-RPs, especially CA11, may play specific roles in the pathogenesis of Machado-Joseph disease.

Mutation of amino acid residues 94, 96 and 119 to histidine(s) in the human carbonic anhydrase (CA, EC 4.2.1.1) related proteins CARP VIII, X and XI restored the zinc binding and catalytic activity for the hydration of CO(2) to bicarbonate. CA VIII, X and XI thus obtained showed high catalytic activity (67.3-92.0% of the activity of hCA II and much higher compared to hCA I) and were inhibited in the milli-micromolar range by inorganic anions, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid. Among the three new isoforms, hCA X was the most efficient enzyme and also showed the highest affinity for anion inhibitors (K(I)s of 3.6-68 μM for phenylboronic acid, sufamic acid, sulfamide, cyanide and azide). hCA VIII was poorly inhibited by halides, cyanate, nitrate and sulfate (K(I)s of 38.4-65.4 mM), whereas CA XI had a behavior intermediate between that of hCA VIII and X, both regarding the catalytic activity and sensitivity to anion inhibitors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were investigated for their inhibition with a large panel of sulfonamides and sulfamates. Unlike inorganic anions, which are weak, millimolar inhibitors of the two enzymes [Vullo et al., Bioorg. Med. Chem. Lett.2011, 21, 3591], sulfonamides and sulfamates are effective micro-to nanomolar inhibitors of the two enzymes. Various types of inhibitors have been detected among the 38 investigated sulfonamides/sulfamates, with K(I)s in the range of 31 nM-5.87 μM. The best stCA 1 inhibitors were acetazolamide and benzolamide-based compounds, whereas the best stCA 2 inhibitors were sulfonylated benzenesulfonamides and amino-benzolamide derivatives (K(I)s in the range of 31-90 nM). 3-Fluoro-5-chloro-4-aminobenzolamide showed an inhibition constant of 51 nM against stCA 1 and of 38 nM against stCA 2, being the best inhibitor detected so far for these enzymes. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing novel antibacterials, targeting β-CAs which may be involved in pathogenicity and invasion of some bacteria., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Two new β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were characterized kinetically. The two enzymes possess appreciable activity as catalysts for the hydration of CO(2) to bicarbonate, with k(cat) of 0.79×10(6) s(-1) and 1.0×10(6) s(-1), and k(cat)/K(m) of 5.2×10(7) M(-1) s(-1) and of 8.3×10(7) M(-1) s(-1), respectively. A large number of simple/complex inorganic anions as well as other small molecules (sulfamide, sulfamic acid, phenylboronic acid, phenylarsonic acid, dialkyldithiocarbamates) showed interesting inhibitory properties towards the two new enzymes, with several low micromolar inhibitors discovered. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing lead compounds for novel types of antibacterials., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A β-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA II, has been cloned, purified, and characterized kinetically. bsCA II showed high catalytic activity for the hydration of CO(2) to bicarbonate, with a k(cat) of 1.1×10(6), and k(cat)/K(m) of 8.9×10(7)M(-1)s(-1). A panel of sulfonamides and sulfamates have been investigated for inhibition of this enzyme. All types of activities, from the low nanomolar to the micromolar, have been detected for these derivatives, which showed inhibition constants in the range of 7.3nM-8.56μM. The best bsCA II inhibitors were some glycosylated sulfanilamides, aliphatic sulfamates, and halogenated sulfanilamides, with inhibition constants of 7.3-87nM. Some of these dual inhibitors of bsCA I and II, also inhibited bacterial growth in vitro, in liquid cultures. These promising data on live bacteria allow us to propose bacterial β-CA inhibition as an approach for obtaining anti-infective agents with a new mechanism of action compared to classical antibiotics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A beta-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA 1, has been cloned, purified, and characterized kinetically. bsCA 1 has appreciable activity as catalyst for the hydration of CO(2) to bicarbonate, with a k(cat) of 6.4 x 10(5) s(-1) and k(cat)/K(m) of 3.9 x 10(7) M(-1).s(-1). A panel of 38 sulfonamides and one sulfamate have been investigated for inhibition of this new beta-CA. All types of activities have been detected, with K(I)s in the range of 17 nM to 5.87 microM. The best bsCA 1 inhibitors were ethoxzolamide (17 nM), celecoxib (18 nM), dorzolamide (21 nM), valdecoxib, and sulpiride (19 nM). Whether bsCA 1 inhibitors may have application in the fight against brucellosis, an endemic disease and the major bacterial zoonosis, producing debilitating infection in humans and animals, warrants further studies.

Three β-carbonic anhydrases (CAs, EC 4.2.1.1), encoded by the gene Rv1284 (mtCA 1) Rv3588c (mtCA 2) and Rv3273 (mtCA 3) are present in the human pathogen Mycobacterium tuberculosis. These enzymes were cloned and they showed appreciable catalytic activity for CO(2) hydration, with k(cat) of 3.9 x 10(5) s(-1), and k(cat)/K(m) of 3.7 x 10(7) M(-1).s1 for mtCA 1, of 9.8 x 10(5) s(-1), and k(cat)/K(m) of 9.3 x 10(7) M(-1).s(-1) for mtCA 2 and k(cat) of 4.3 x 10(5) s(-1), and a k(cat)/K(m) of 4.0 x 10(7) M(-1).s(-1) for mtCA 3, respectively. The Rv3273 gene product is predicted to be a 764 amino acid residues polypeptide, consisting of a sulfate transporter domain (amino acids 121-414) in addition to the β-CA mentioned above (which is encoded by residues 571-741). All these enzymes were inhibited appreciably by many sulfonamides and sulfamates, in the nanomolar - micromolar range, whereas some subnanomolar inhibitors were also reported for two of them (mtCA 1 and mtCA 3). As sulfonamides also efficiently inhibit dehydropteroate synthetase (DHPS), the contribution of mtCAs and DHPS inhibition to a possible antimycobacterial action of these drugs must be better understood. It has been, however, proven that mtCAs are druggable targets, with a real potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug resistance and extensive multi-drug resistance, although for the moment no in vivo inhibition of the bacteria could be evidenced with the presently available drugs due to lack of penetrability through the mycolic acid cell wall of M. tuberculosis.

The Rv3273 gene product of Mycobacterium tuberculosis, a beta-carbonic anhydrase (CA, EC 4.2.1.1), mtCA 3, shows appreciable catalytic activity for CO(2) hydration (k(cat) of 4.3 x 10(5) s(-1), and k(cat)/K(m) of 4.0 x 10(7) M(-1) x s(-1)). A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 3. Sulfanilyl-sulfonamides, acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and zonisamide, showed effective, submicromolar inhibition (K(I)s of 104-611 nM), the best inhibitor being 2-amino-pyrimidin-4-yl-sulfanilamide (K(I) of 91 nM).

The beta-carbonic anhydrase (CA, EC 4.2.1.1) encoded by the gene Rv1284 (mtCA 1) of Mycobacterium tuberculosis shows appreciable catalytic activity for CO(2) hydration, with a k(cat) of 3.9 x 10(5) s(-1) and a k(cat)/K(m) of 3.7 x 10(7) M(-1) s(-1). A panel of 36 sulfonamides and one sulfamate, some of which are used clinically, were assayed for their effect on mtCA 1 catalytic activity. Most sulfonamides exhibited K(I) values in the range of 1-10 microM, but several derivatives, including sulfanilyl-sulfonamides acetazolamide, methazolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and the sulfamate topiramate, exhibited submicromolar inhibition (K(I) values of 0.481-0.905 microM). The best inhibitors were 3-bromosulfanilamide and indisulam (K(I) values of 97-186 nM). This study demonstrates that mtCA 1 can be inhibited by sulfonamides and sulfamates and thus has potential for developing antimycobacterial agents with an alternate mechanism of action. This is an important finding to explore further, as many strains exhibit multidrug resistance and extensive multidrug resistance to existing therapeutics.

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(- 1) and k(cat)/K(M) of 2.5.10(5) M(- 1) s(- 1). The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (K(I)s of 21-78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed K(I)s around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed K(I)s in the range of 10-90 microM. It is difficult to explain the inhibitory activity of carbonate (K(I) of 10 microM) against hCA III, also considering the fact that this ion has an affinity of 15-73 mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (K(I) of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(-1) and k(cat)/K(M) of 2.5 x 10(5) M(-1) s(-1), being a slower catalyst for the physiological reaction as compared to the genetically related cytosolic isoforms hCA I and II. An inhibition study with a library of sulfonamides and one sulfamate, some which are clinically used compounds, is reported. hCA III is less prone to be inhibited by these compounds as compared to hCA I and II for which many low nanomolar inhibitors were detected earlier. The best hCA III inhibitors were prontosil, sulpiride, indisulam, benzolamide, aminobenzolamide, and 4-amino-6-chloro-benzene-1,3-disulfonamide which showed K(I)s in the range of 2.3-18.1 microM. Clinically used compounds such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, topiramate, zonisamide, celecoxib, and valdecoxib were less effective hCA III inhibitors, with affinities in the range of 154-2200 microM. This is the first study in which low micromolar hCA III inhibitors are reported.

DNA clones for the beta-class carbonic anhydrase (CA, EC 4.2.1.1) of Helicobactor pylori (hpbetaCA) were obtained. A recombinant hpbetaCA protein lacking the N-terminal 15-amino acid residues was produced and purified, representing a catalytically efficient CA. hpbetaCA was strongly inhibited (K(I)s in the range of 24-45 nM) by many sulfonamides/sulfamates, among which acetazolamide, ethoxzolamide, topiramate, and sulpiride, all clinically used drugs. The dual inhibition of alpha- and/or beta-class CAs of H. pylori might represent a useful alternative for the management of gastritis/gastric ulcers, as well as gastric cancer. This is also the first study showing that a bacterial beta-CA can be a drug target.

Increased expression of carbonic anhydrase-related protein (CA-RP) VIII has previously been shown in colorectal carcinoma. Since CA-RP has no catalytic carbonic anhydrase (CA) activity, the present study attempted to elucidate its biological significance in colon cancer cells. From a colon cancer cell line (LoVo), we established clones that overexpressed CA-RP VIII (LoVo-CA8) and a control transfectant with a vector alone (LoVo-pCIneo) and studied alterations in the biological behaviors of the tumor cells both in vitro and in vivo. LoVo-CA8 cells showed significantly increased mRNA and protein expressions of CA-RP VIII as compared to LoVo-pCIneo cells. Cell proliferation, colony formation, and cell invasion assays showed that LoVo-CA8 cells had significantly higher cell proliferative and invasive abilities as compared to parental LoVo and LoVo-pCIneo cells in vitro. In an in vivo xenograft assay, LoVo-CA8 cells showed a higher tumor growth rate than parental LoVo cells. Further, small interfering RNA (siRNA)-mediated knockdown of CA-RP VIII revealed significant inhibition in cell proliferation and colony formation of a colon cancer cell line HCT116, which showed high endogenous expression of CA-RP VIII. These findings indicated that CA-RP VIII plays a role in the growth of colon cancer cells., ((c) 2007 Wiley-Liss, Inc.)

The secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified in a bacterial expression system. The kinetic parameters for the CO2 hydration reaction proved hCA VI to possess a kcat of 3.4 x 10(5) s-1 and kcat/KM of 4.9 x 10(7) M-1 s-1 (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction on the same order of magnitude as the ubiquitous isoform CA I or the transmembrane, tumor-associated isozyme CA IX. A series of sulfonamides and one sulfamate have been tested for their interaction with this isozyme. Simple benzenesulfonamides were rather ineffective hCA VI inhibitors, with inhibition constants in the range of 1090-6680 nM. Better inhibitors were detected among such derivatives bearing 2- or 4-amino-, 4-aminomethyl-, or 4-hydroxymethyl moieties or among halogenated sulfanilamides (KI values of 608-955 nM). Some clinically used compounds, such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, sulpiride, and indisulam, or the orphan drug benzolamide, showed effective hCA VI inhibitory activity, with inhibition constants of 0.8-79 nM. The best inhibitors were brinzolamide and sulpiride (KI values of 0.8-0.9 nM), the latter compound being also a CA VI-selective inhibitor. The metallic taste reported as a side effect after the treatment with systemic sulfonamides may be due to the inhibition of the salivary CA VI. Some of the compounds investigated in this study might be used as additives in toothpastes for reducing the acidification produced by the relevant CO2 hydrase activity of enamel CA VI, which leads to the formation of protons and bicarbonate and may have a role in cariogenesis.

Solid-pseudopapillary tumor (SPT) is an unusual pancreatic neoplasm that is characterized by a mixture of solid and cystic components and a fibrous capsule. Recently, the tumorigenesis of SPT has been reported to be associated with gene mutations of beta-catenin, which is a molecule participating in the Wnt signaling pathway. Reported herein is the case of a 53-year-old woman with SPT. The tumor, approximately 3 cm in diameter in the pancreas body, had a clear margin and central calcification but had neither a cystic component nor fibrous capsule. Several lines of pathological findings in the surgically resected specimen indicated SPT: (i) pseudopapillary proliferation of eosinophilic polygonal cells with oval nuclei; (ii) positive expression of several marker molecules indicating differentiation into acinar and endocrine cells; and (iii) zymogen granule-like structures in the cytoplasm on electron microscopy. Further, the tumor cells had intense nuclear accumulation of beta-catenin and an activating mutation, (34)Gly(GGA) to Arg(AGA), in exon 3 of the beta-catenin gene, as previously reported in most SPT. These findings suggest that association of the beta-catenin phenotype with development of the rare phenotype of SPT, a non-cystic and unencapsulated tumor, is unlikely.

Carbonic anhydrase-related protein VIII (CA-RP VIII) is believed to be an oncofetal antigen and is overexpressed in colorectal and non-small cell lung cancer. However, the pathobiological properties of CA-RP VIII in lung cancer remain unclear. In the present study, we examined ultrastructural changes caused by exogenous CA-RP VIII expression in a well-differentiated lung adenocarcinoma cell line, PC-9. Many vacuoles lined by cilia, sometimes large vacuoles pushing the nuclei to one side, were found in the cytoplasm of CA-RP VIII-expressing PC-9 cells, but not in control PC-9 cells. Moreover, signet-ring cells containing abundant intracytoplasmic mucin were often found among CA-RP VIII-expressing PC-9 cells, but rarely among control PC-9 cells. We subsequently examined CA-RP VIII expression in atypical adenomatous hyperplasia and early-stage lung adenocarcinoma (Stage Ia). Significant expression of CA-RP VIII was observed in invasive lung adenocarcinoma but not in noninvasive adenocarcinoma. Interestingly, CA-RP VIII was strongly expressed in signet-ring cell cancer and invasive mucinous adenocarcinoma components. CA-RP VIII also appeared to enhance the invasiveness of PC-9 cells in Matrigel invasion assay. The present findings suggest that CA-RP VIII expression in lung adenocarcinoma is related to cancer cell invasion.

A library of sulfonamides/sulfamates has been investigated for the inhibition of the carboxyterminal truncated form of the alpha-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the gastric pathogen Helicobacter pylori (hpCA). This enzyme, incorporating 202 amino acid residues, showed a catalytic activity similar to that of the full length hpCA, with k(cat) of 2.35 x 10(5)s(-1) and k(cat)/K(M) of 1.56 x 10(7)M(-1)s(-1) at 25 degrees C and pH of 8.9, for the CO(2) hydration reaction. All types of activity for inhibition of the bacterial enzyme have been detected. Dorzolamide and simple 4-substituted benzenesulfonamides were weak hpCA inhibitors (inhibition constants, K(I)s, in the range of 830-4310 nM). Sulfanilamide, orthanilamide, some of their derivatives, and indisulam showed better activity (K(I)s in the range of 310-562 nM), whereas most of the clinically used CA inhibitors, such as methazolamide, ethoxzolamide, dichlorophenamide, brinzolamide, topiramate, zonisamide, etc., acted as medium potency hpCA inhibitors (K(I)s in the range of 124-287 nM). Some potent hpCA inhibitors were detected too (K(I)s in the range of 20-96 nM) such as acetazolamide, 4-amino-6-chloro-1,3-benzenedisulfonamide, 4-sulfanilyl-aminoethyl-benzenesulfonamide, and 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. Most of the investigated derivatives acted as better inhibitors of the human isoform hCA II than as hpCA inhibitors. Since hpCA is essential for the survival of the pathogen in acid, its inhibition by compounds such as those investigated here might be used as a new pharmacologic tool in the management of drug resistant H. pylori.

We have cloned and sequenced Helicobacter pylori alpha-class carbonic anhydrase (hpCA) from patients with different gastric mucosal lesions, including gastritis (n=15), ulcer (n=6), and cancer (n=16). Although several polymorphisms were newly identified such as 12Ala, 13Thr, 16Ile, and 168Phe, there was no significant relevance of any polymorphism with gastric mucosal lesion types. A library of sulfonamides/sulfamates has been investigated for the inhibition of hpCA, whereas new derivatives have been obtained by attaching 4-tert-butyl-phenylcarboxamido/sulfonamido tails to benzenesulfonamide/1,3,4-thiadiazole-2-sulfonamide scaffolds. All types of activity for inhibition of hpCA have been detected. Dorzolamide and simple 4-substituted benzenesulfonamides were weak inhibitors (KI 873-4360 nM). Sulfanilamide, orthanilamide, some of their derivatives, and indisulam showed better activity (KI 413-640 nM), whereas most of the clinically used inhibitors, such as methazolamide, ethoxzolamide, dichlorophenamide, brinzolamide, topiramate, zonisamide, etc., acted as medium-potency inhibitors (KI 105-378 nM). Some potent hpCA inhibitors were detected too (KI 12-84 nM) among acetazolamide, 4-amino-6-chloro-1,3-benzenedisulfonamide and some newly designed compounds incorporating lipophilic tails. Some of the newly prepared derivatives had selectivity ratios for inhibiting hpCA over hCA II in the range of 1.25-3.48, showing thus some selectivity for inhibiting the bacterial enzyme. Since hpCA is essential for the survival of the pathogen in acid, it might be used as a new pharmacologic tool in the management of drug-resistant H. pylori.

The methanolic extract of Myrsine seguinii yielded three anti-inflammatory compounds, myrsinoic acids B, C and F, and their structures were elucidated from the spectroscopic data. These compounds suppressed the TPA-induced edema of mouse ear, myrsinoic acid F being the most active (IE 77% at a dose of 0.56 micromol).

We analysed 267 samples from patients with lung cancer and 54 samples from patients with pancreatic cancer and classified them into three types (A, B and C) based on their P450CYPIA1 genotypes. This was performed by restriction fragment length polymorphism (RFLP) analysis of DNA (that had been amplified) using the polymerase chain reaction (PCR). Our results reconfirmed previous studies with RFLP analyses by revealing significantly higher frequencies of C type in lung cancer patients, particularly light smokers with squamous cell carcinoma (p < 0.001). Also when we compared the appearance rate of P450CYPIA1 genotype in lung cancer patients between metastasis group and no metastasis group, lymph node or distant metastasis was more frequently observed among the patients of squamous cell carcinoma, especially the light smoking group, with C genotypes (p < 0.05). These results suggested that this genotype is not only associated with the etiology of lung cancer but also with the status of metastasis. Furthermore, genotyping could be useful in predicting the incidence of lung cancer in the smokers and the general population as well as the prognosis of individuals who develop this disease.