The proteoglycan region of the tumor-associated carbonic anhydrase isoform IX acts as anintrinsic buffer optimizing CO2 hydration at acidic pH values characteristic of solid tumors

The enzymatic activities of carbonic anhydrase (CA, EC 4.2.1.1) isozymes CA I, II, IX (catalytic domain (cdCA IX) and catalytic domain plus proteoglycan, flCA IX), XII and XIV were investigated as a function of pH for the CO₂ hydration to bicarbonate and a proton. The cytosolic isoforms CA I and II as well as the catalytic domain of CA IX, together with the transmembrane isoforms CA XII and XIV showed sigmoid pH dependencies of k_cat/K_M, with a pK_a of 6.90–7.10, showing thus optimal catalytic efficiency around pH 7. The full length CA IX had a similar shape of the pH dependency curve but with a pK_a of 6.49, having thus maximal catalytic activity at pH values around 6.5, typical of hypoxic solid tumors in which CA IX is overexpressed. The proteoglycan domain of CA IX (present only in this transmembrane isoform) may thus act as an intrinsic buffer promoting efficient CO₂ hydration at acidic pH values found in hypoxic tumors.     

Carbonic anhydrase inhibitors. Inhibition of the fungal β-carbonic anhydrases from Candida albicans and Cryptococcus neoformans with boronic acids

Inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with a series of aromatic, arylalkenyl- and arylalkylboronic acids was investigated. Aromatic, 4-phenylsubstituted- and 2-naphthylboronic acids were the best Can2 inhibitors, with inhibition constants in the range of 8.5–11.5 μM, whereas arylalkenyl and aryalkylboronic acids showed K_Is in the range of 428–3040 μM. Nce103 showed a similar inhibition profile, with the 4-phenylsubstituted- and 2-naphthylboronic acids possessing K_Is in the range of 7.8–42.3 μM, whereas the arylalkenyl and aryalkylboronic acids were weaker inhibitors (K_Is of 412–5210 μM). The host human enzymes CA I and II were also effectively inhibited by these boronic acids. The B(OH)₂ moiety is thus a new zinc-binding group for designing effective inhibitors of the α- and β-CAs.     

Carbonic anhydrase inhibitors: The membrane-associated isoform XV is highly inhibited by inorganic anions

The membrane-associated mouse isozyme of carbonic anhydrase XV (mCA XV), has been investigated for its interaction with anion inhibitors. mCA XV is an isoforms possessing a very particular inhibition profile by anions, dissimilar to that of all other mammalian CAs investigated earlier. Many simple inorganic anions (thiocyanate, cyanide, azide, bicarbonate, hydrogen sulfide, bisulfite and sulfate) showed low micromolar inhibition constants against mCA XV (K_Is of 8.2–10.1 μM), whereas they acted as much weaker (usually millimolar) inhibitors of other isoforms. Halides, nitrate, nitrite, carbonate, sulfamate, sulfamide and phenylboronic/arsonic acid were weaker inhibitors, with inhibition constants in the range of 27.6–288 μM. Our data may be useful for the design of more potent inhibitors of mCA XV (considering various zinc binding groups present in the anions investigated here, e.g., the sulfonate one) and for understanding some physiologic/pharmacologic consequences of mCA XV inhibition by anions such as bicarbonate or sulfate which show quite high affinity for it.     

Carbonic anhydrase inhibitors. The nematode α-carbonic anhydrase of Caenorhabditis elegans CAH-4b is highly inhibited by 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides

A series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides possessing various 2-, 3- or 4-substituted phenyl groups with methyl-, halogeno- and methoxy-functionalities, as well as the perfluorophenyl moiety, have been evaluated as inhibitors of an α-carbonic anhydrase (CA, EC 4.2.1.1) of the nematode model organism Caenorhabditis elegans (CAH-4b, or ceCA). The substitution pattern at the 3-phenyl ring highly influenced the ceCA inhibitory activity of these heterocyclic sulfonamides, with best inhibitors (K_Is in the range of 6.0–13.4 nM) incorporating 3-methyl-, 4-methyl-, 2-/3-/4-fluoro-, 4-chloro- and 3-/4-bromo-phenyl such moieties. Some of these sulfonamides also showed a good selectivity profile for the inhibition of the nematode over the human isozymes CA I and II (selectivity ratios in the range of 1.78–4.95 for the inhibition of ceCA over hCA II). These data can be used for the design of possibly new antihelmintic drugs, since the genome of many parasitic nematodes encode for a multitude of orthologue CA isozymes to ceCA investigated here.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with aliphatic and aromatic carboxylates

The inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with carboxylates such as the C1–C5 aliphatic carboxylates, oxalate, malonate, maleate, malate, pyruvate, lactate, citrate and some benzoates has been investigated. The best Can2 inhibitors were acetate and maleate (K_Is of 7.3–8.7 μM), whereas formate, acetate, valerate, oxalate, maleate, citrate and 2,3,5,6-tetrafluorobenzoate showed less effective inhibition, with K_Is in the range of 42.8–88.6 μM. Propionate, butyrate, malonate, l-malate, pyruvate, l-lactate and benzoate, were weak Can2 inhibitors, with inhibition constants in the range of 225–1267 μM. Nce103 was more susceptible to inhibition with carboxylates compared to Can2, with the best inhibitors (maleate, benzoate, butyrate and malonate) showing K_Is in the range of 8.6–26.9 μM. l-Malate and pyruvate together with valerate were the less efficient Nce103 inhibitors (K_Is of 87.7–94.0 μM), while the remaining carboxylates showed a compact behavior of efficient inhibitors (K_Is in the range of 35.1–61.6 μM). Notably the inhibition profiles of the two fungal β-CAs was very different from that of the ubiquitous host enzyme hCA II (belonging to the α-CA family), with maleate showing selectivity ratios of 113.6 and 115 for Can2 and Nce103, respectively, over hCA II inhibition. Therefore, maleate is a promising starting lead molecule for the development of better, low nanomolar, selective β-CA inhibitors.     

Distribution and coexistence of shrews in patchy landscapes: A field test of multiple hypotheses

Despite the important role of shrews (Soricomorpha: Soricidae) in the functioning of ecosystems, as predators and prey, the effects of habitat loss and fragmentation on this guild of mammals are still unclear. We studied the distribution of 5 species (the greater white toothed shrew Crocidura leucodon; the lesser white toothed shrew Crocidura suaveolens; the pigmy shrew Sorex minutus; the Appennine shrew Sorex samniticus and the Etruscan shrew Suncus etruscus) in a fragmented landscape in central Italy.Shrews were trapped with pitfall traps made from plastic water bottles, the number of traps increased with patch size. A total of 170 individuals, of 5 species of shrews were captured. Shrews were widely distributed in our study area, however patch occupancy was determined mainly by vegetation and geometrical characteristics of the patches. Our data supports the hypotheses that patterns of habitat selection and the dynamics of seasonal abundance (habitat and temporal partitioning between similarly sized species) reduce competitive pressure, thus allowing coexistence of shrews in relatively species-rich assemblages, for such small amounts of habitat. The most important outcome of our results is the crucial role played by vegetation structure in determining distribution patterns. These results strongly suggest that measurements of the vegetation structure of habitat patches should always be included as explanatory variables when studying the distribution of shrews in fragmented landscapes.     

Tetramerization and Cooperativity in Plasmodium falciparum Glutathione S-Transferase Are Mediated by Atypic Loop 113�C119

Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modulated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113–119 in the tetramerization process and in the cooperative phenomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoenzymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST, it could be a new target for the development of antimalarial drugs.Approximately two million deaths in the world per year are caused by Plasmodium falciparum, the parasite responsible for tropical malaria (1, 2). In the last years, increasing interest has been developing for the peculiar glutathione S-transferase (PfGST)³ expressed by this parasite. Expressed in almost all living organisms, GSTs represent a large superfamily of multifunctional detoxifying enzymes that are able to conjugate GSH to a lot of toxic electrophilic compounds, thus facilitating their excretion. Many other protection roles of GSTs have been described, including the enzymatic reduction of organic peroxides (3–5), the inactivation of the proapoptotic JNK through a GST·JNK complex (6), and the protection of the cell from excess nitric oxide (7). The mammalian cytosolic GSTs are dimeric proteins grouped into eight species-independent classes termed Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta on the basis of sequence similarity, immunological reactivity, and substrate specificity (3, 8–11). PfGST is one of the most abundant proteins expressed by P. falciparum (from 1 to 10%, i.e. from 0.1 to 1 mm) (12), and different from what occurs in many organisms, it is the sole GST isoenzyme expressed by this parasite. Despite its structural similarity to the Mu class GST, this specific isoenzyme cannot be assigned to any known GST class (13). The interest in this enzyme is due to its particular protective role in the parasite. In fact, in addition to the usual GST activity that promotes the conjugation of GSH to electrophilic centers of toxic compounds, this protein efficiently binds hemin, and thus it could protect the parasite (that resides in the erythrocytes) from the parasitotoxic effect of this heme by-product (14). Specific compounds that selectively inhibit its catalytic activity or hemin binding could be promising candidates as antimalarial drugs. In this context, the discovery of structural or mechanistic properties of this enzyme that are not found in other GSTs may be important for designing selective inhibitors that are toxic to the parasite but harmless for the host cells. Two properties never observed in other members of the GST superfamily are of particular interest. The first property is that this enzyme, in the absence of GSH, is inactivated in a short time and loses its ability to bind hemin (15). Recent studies indicated that the inactivation process is related to a dimer to tetramer transition (13, 16, 17). The second property is the strong positive homotropic phenomenon that modulates the affinity of the two subunits for hemin (15). The x-ray crystal structure of PfGST, solved by two different groups (13, 18), provides insights into this effect. From a structural point of view, the most intriguing differences of PfGST when compared with other GSTs are a more solvent-exposed H-site and an atypic extra loop connecting helix α-4 and helix α-5 (residues 113–119; see also Fig. 1) that could be involved in the dimer-dimer interaction. Actually, in the absence of ligands, two biological dimers form a tetramer, and these homodimers are interlocked with each other by loop 113–119 of one homodimer, which occupies an H-site of the other homodimer (13, 18). Upon binding of S-hexylglutathione, loop 113–119 rearranges; residues Asn-114, Leu-115, and Phe-116 form an additional coil in helix α-4; and the side chains of Asn-111, Phe-116, and Tyr-211 flip into the H-site of the same dimer (17, 18). The changed course of residues 113–119 in the liganded enzyme prevents the interlocking of the dimers.Open in a separate windowFIGURE 1.A, structural changes of loop 113–119 occurring in the dimer (light blue model and yellow loop; Protein Data Bank code 2AAW) to tetramer (blue model and orange loop; Protein Data Bank code 1OKT) transition. Red spheres indicate the amino acids replaced in this study to obtain mutants A, B, and C. B, model of hemin·PfGST complex obtained by docking simulation using the crystal structure for Protein Data Bank code 1Q4J (15). Hemin is shown in red, loop 113–119 is in orange, and GSH is shown as yellow sticks.In this paper, by means of site-directed mutagenesis, fluorescence anisotropy, kinetic studies, and size exclusion chromatography, we check the influence of selected mutations of this atypic loop in the tetramerization process and the possible involvement of this protein segment in the cooperative phenomenon that characterizes hemin binding. In addition we describe that the tetramerization process is inhibited not only by GSH but even by GSSG. This finding suggests that hemin binding of PfGST is independent of the redox status of the cell. Finally, we demonstrate that the presence of GSH (or GSSG) in the active site is not essential for hemin binding, but this interaction only requires an active dimeric conformation.     

Effects of TMS on Different Stages of Motor and Non-Motor Verb Processing in the Primary Motor Cortex

The embodied cognition hypothesis suggests that motor and premotor areas are automatically and necessarily involved in understanding action language, as word conceptual representations are embodied. This transcranial magnetic stimulation (TMS) study explores the role of the left primary motor cortex in action-verb processing. TMS-induced motor-evoked potentials from right-hand muscles were recorded as a measure of M1 activity, while participants were asked either to judge explicitly whether a verb was action-related (semantic task) or to decide on the number of syllables in a verb (syllabic task). TMS was applied in three different experiments at 170, 350 and 500 ms post-stimulus during both tasks to identify when the enhancement of M1 activity occurred during word processing. The delays between stimulus onset and magnetic stimulation were consistent with electrophysiological studies, suggesting that word recognition can be differentiated into early (within 200 ms) and late (within 400 ms) lexical-semantic stages, and post-conceptual stages. Reaction times and accuracy were recorded to measure the extent to which the participants'' linguistic performance was affected by the interference of TMS with M1 activity. No enhancement of M1 activity specific for action verbs was found at 170 and 350 ms post-stimulus, when lexical-semantic processes are presumed to occur (Experiments 1–2). When TMS was applied at 500 ms post-stimulus (Experiment 3), processing action verbs, compared with non-action verbs, increased the M1-activity in the semantic task and decreased it in the syllabic task. This effect was specific for hand-action verbs and was not observed for action-verbs related to other body parts. Neither accuracy nor RTs were affected by TMS. These findings suggest that the lexical-semantic processing of action verbs does not automatically activate the M1. This area seems to be rather involved in post-conceptual processing that follows the retrieval of motor representations, its activity being modulated (facilitated or inhibited), in a top-down manner, by the specific demand of the task.     

Motor Properties of Peripersonal Space in Humans

Background

A stimulus approaching the body requires fast processing and appropriate motor reactions. In monkeys, fronto-parietal networks are involved both in integrating multisensory information within a limited space surrounding the body (i.e. peripersonal space, PPS) and in action planning and execution, suggesting an overlap between sensory representations of space and motor representations of action. In the present study we investigate whether these overlapping representations also exist in the human brain.

Methodology/Principal Findings

We recorded from hand muscles motor-evoked potentials (MEPs) induced by single-pulse of transcranial magnetic stimulation (TMS) after presenting an auditory stimulus either near the hand or in far space. MEPs recorded 50 ms after the near-sound onset were enhanced compared to MEPs evoked after far sounds. This near-far modulation faded at longer inter-stimulus intervals, and reversed completely for MEPs recorded 300 ms after the sound onset. At that time point, higher motor excitability was associated with far sounds. Such auditory modulation of hand motor representation was specific to a hand-centred, and not a body-centred reference frame.

Conclusions/Significance

This pattern of corticospinal modulation highlights the relation between space and time in the PPS representation: an early facilitation for near stimuli may reflect immediate motor preparation, whereas, at later time intervals, motor preparation relates to distant stimuli potentially approaching the body.