Cloning,characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii

We investigated the catalytic activity and inhibition of the δ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii, TweCA. The enzyme, obtained by cloning the synthetic gene, was an efficient catalyst for the CO₂ hydration, its physiological reaction, with a k_cat of 1.3 × 10⁵ s⁻¹ and a k_cat/K_M of 3.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (K_Is >200 mM) whereas other halides and pseudohalides were submillimolar–millimolar inhibitors (K_Is in the range of 0.93–8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 9–90 μM, whereas acetazolamide inhibited the enzyme with a K_I of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. However, these enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO₂ fixation by sea organisms. A phylogenetic analysis with all five genetic families of CAs showed that α- and δ-CAs are evolutionarily more related to each other with respect to the γ-CAs, although these three families clustered all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and their potential biotechnological applications in biomimetic CO₂ capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms.     

Inhibition of the R1 fragment of the cadmium-containing ζ-class carbonic anhydrase from the diatom Thalassiosira weissflogii with anions

We investigated the catalytic activity and inhibition of both the zinc and cadmium-containing R1 fragment of the ζ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii. Our data prove that these enzymes are not only very efficient catalysts for the physiological reaction, but also sensitive to sulfonamide and anion inhibitors, with inhibition constants from the nanomolar to millimolar range. Acetazolamide inhibited the two enzymes with K_Is in the range of 58–92 nM. The best anion inhibitors of Cd-R1 were thiocyanate, sulfamate and sulfamide, with K_Is of 10–89 μM, whereas the best Zn-R1 anion inhibitors were sulfamate and sulfamide with K_Is of 60–72 μM. These enzymes were only weakly inhibited by chloride, bromide or sulfate, main anion components of sea water, with inhibition constants in the range of 0.24–0.85 mM. Thus, similarly to CAs belonging to other classes, the ζ-class CA (with either cadmium or zinc ions at the active site) was inhibited by both anions and sulfonamides.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzyme from the pathogenic yeast Candida glabrata with anions

A β-carbonic anhydrase (CA, EC 4.2.1.1), the protein encoded by the NCE103 gene of Candida glabrata which also present in Candida albicans and Saccharomyces cerevisiae, was cloned, purified, characterized kinetically and investigated for its inhibition by a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate and some isosteric species. The enzyme showed significant CO₂ hydrase activity, with a k_cat of 3.8 × 10⁵ s^?1 and k_cat/K_M of 4.8 × 10⁷ M^?1 s^?1. The Cà glabrata CA (CgCA) was moderately inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K_Is of 0.60–1.12 mM) but strongly inhibited by bicarbonate, nitrate, nitrite and phenylarsonic acid (K_Is of 86–98 μM). The other anions investigated showed inhibition constants in the low millimolar range, with the exception of bromide and iodide (K_Is of 27–42 mM).     

Carbonic anhydrase inhibitors: Inhibition of the β-class enzyme from the yeast Saccharomyces cerevisiae with sulfonamides and sulfamates

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically and investigated for its inhibition with a series of sulfonamides and one sulfamate. The enzyme showed high CO₂ hydrase activity, with a k_cat of 9.4 × 10⁵ s^?1, and k_cat/K_M of 9.8 × 10⁷ M^?1 s^?1. Simple benzenesulfonamides substituted in 2-, 4- and 3,4-positions of the benzene ring with amino, alkyl, halogeno and hydroxyalkyl moieties were weak scCA inhibitors with K_Is in the range of 0.976–18.45 μM. Better inhibition (K_Is in the range of 154–654 nM) was observed for benzenesulfonamides incorporating aminoalkyl/carboxyalkyl moieties or halogenosulfanilamides; benzene-1,3-disulfonamides; simple heterocyclic sulfonamides and sulfanilyl-sulfonamides. The clinically used sulfonamides/sulfamate (acetazolamide, ethoxzolamide, methazolamide, dorzolamide, topiramate, celecoxib, etc.) generally showed effective scCA inhibitory activity, with K_Is in the range of 82.6–133 nM. The best inhibitor (K_I of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. These inhibitors may be useful to better understand the physiological role of β-CAs in yeast and some pathogenic fungi which encode orthologues of the yeast enzyme and eventually for designing novel antifungal therapies.     

Notes on the genus Clematis (Ranunculaceae) (Ⅱ)

(1) The systematic positions of Clematis potaninii Maxim., C . heynei Rau, C. trichotoma Nakai, C. apiculata Hook. f. & Thoms, C. theobromina Dunn, C. sigensis Engler,C. hedysarifolia DC., and C. dissecta Baker, and the specific status of C. trifida Hook, C.pimpinellifolia Hook., C. oligophylla Hook., and Clematopsis lineariloba Hutch. are discussed; (2) New classifications for Clematis parviloba Gardn. & Champ., C. puberula Hook. f. & Thoms., and sect. Naraveliopsis Hand.-Mazz. are provided; (3) Clematis subsect. Potaninianae M. Johnson, C. heynei M. Johnson, C. petelotii Gagnep. and some other names are reduced to synonymy; (4) Two subsections, 4 series, 4 species, and 4 varieties are described as new; (5)Four new ranks and 4 new combinations are made.     

Notes on the genus Clematis (Ranunculaceae) (Ⅳ)

(1) Clematis taiwaniana Hayata, C. grata Wall. var. ryukiuensis Tamura, C . sikkimensis (Hook. f. & Thoms. ) Drumm. ex Burkill, C. connata DC. var. bipinnata M. Y. Fang, C. kilungensis W. T. Wang & M. Y. Fang etc. are reduced to synonymy. (2) New classifications for sect. Tubulosae Decne., C. siamensis Drumm. & Craib and C. connata DC. are proposed. (3) 3 series, 3 species and 4 varieties are described as new. (4) 6 new combinations and 3 newranks are made.     

Notes on the genus Clematis (Ranunculaceae) (Ⅲ)

(1) The evolutionary trends of sepals and stamens of the genus Clematis are discussed; (2) New classifications for sect. Cheiropsis DC. and sect. Aspidanthera Spach are proposed; (3) One subsection, 13 series, 5 species and 4 varieties are described as new; (4) Six new ranks and2 new combinations are made.     

Notes on the genus Clematis (Ranunculaceae) (Ⅰ)

( 1 ) Some taxonomical problems of the genus Clematis mainly about misidentifications are discussed, and some treatments including the reinstatement of Clematis montana var. brevifoliola Kuntze, C. apiifolia var. biternata Makino, C. subumbellata Kurz, C. goudotiana Planch. & Triana, C. insidiosa Baill., C. kockiana Schneid. and C. longicauda A. Rich., and the reduction of subsect. Africanae M. Johnson, C. umbellifera Gagnep., C. pubescens Benth., C. rhodocarpa Rose, C. edentata Baker, C. stoltzi Engler, C. tibetana ssp. vernayi var. dentata Grey-Wilson, C. clarkeana var. stenophylla Hand.-Mazz., C. subfalcata Pei ex M.Y. Fang, C. angustifoliola W. T. Wang, C. dasyandra var. polyantha Finet & Gagnep. etc are given. (2) The new diagnoses for the two subsections of the sect. Meclatis are provided; C. sericea H.B. K. ex DC. and C. grossa Benth. are treated as two varieties of one species; and a new classification of the infraspecific taxa of C. hirsuta Perr & Guill. is made. (3) one subsection, one series, eight species and one variety are described as new. (4) The new occurrences of C. montana var. brevifoliola Kuntze in southern Xizang, China, Nepal, Bhutan and northern Myanmar, C. burmanica Lace in southwestern Yunnan, C. armandii Franch. in Assam, India and northern Myanmar,and C. yui W. T. Wang in northern Myanmar are reported.     

Dihalogenated sulfanilamides and benzolamides are effective inhibitors of the three β-class carbonic anhydrases from Mycobacterium tuberculosis

A series of halogenated sulfanilamides and halogenated benzolamide derivatives have been investigated as inhibitors of three β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Mycobacterium tuberculosis, mtCA 1 (Rv1284), mtCA 2 (Rv3588c) and mtCA 3 (Rv3273). All three enzymes were inhibited with efficacies between the submicromolar to the micromolar one, depending on the substitution pattern at the sulfanilamide moiety/fragment of the molecule. Best inhibitors were the halogenated benzolamides (K_Is in the range of 0.12–0.45 μM) whereas the halogenated sulfanilamides were slightly less inhibitory (K_Is in the range of 0.41–4.74 μM). This class of β-CA inhibitors may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug/extensive multi-drug resistance.     

Effect of the alkaline cations on the stability of the model polynucleotide poly(dG-dC)·poly(dG-dC)

When the model polynucleotide poly(dG-dC)?poly(dG-dC) [polyGC] is titrated with a strong acid (HCl) in unbuffered aqueous solutions containing the chlorides of the alkali metals in the concentration range 0.010?M-0.600?M, two transitions in the absorbance vs. pH plots are evidenced, characterized by the constants pK(a(?)) and pK(a(?)). The limiting values at infinite saline concentrations of these two constants, namely pK(∞)(a(?)) and pK(∞)(a(?)) obtained making use of the "one site saturation constant" equation or, in turn, of the double logarithmic plot: pK(a) vs. log([salt]?1), exhibit a clear dependence on the nature of the cations. The effects of the different alkali cations on the pK(∞)(a) values follow the Hofmeister series. In fact, the pK(∞)(a(?)) and the pK(∞)(a(?)) values are smaller for Li+ and Na+ than for Rb+ and Cs+, with K+ at the border between the two, showing that the transitions require higher concentrations of protons to occur in the presence of high concentrations of the cosmotropic ions.     

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.     

Inhibition survey with phenolic compounds against the δ- and η-class carbonic anhydrases from the marine diatom thalassiosira weissflogii and protozoan Plasmodium falciparum

Abstract

The inhibition of δ- and η-class carbonic anhydrases (CAs; EC 4.2.1.1) was poorly investigated so far. Only one δ-CA, TweCA from the diatom Thalassiosira weissflogii, and one η-CA, PfCA, from Plasmodium falciparum, have been cloned and characterised to date. To enrich δ- and η-CAs inhibition profiles, a panel of 22 phenols was investigated for TweCA and PfCA inhibition. Some derivatives showed effective, sub-micromolar inhibition of TweCA (K_Is 0.81–65.4?µM) and PfCA (K_Is 0.62–78.7?µM). A subset of compounds demonstrated a significant selectivity for the target CAs over the human physiologically relevant ones. This study promotes the identification of new potent and selective inhibitors of TweCA and PfCA, which could be considered as leads for finding molecular probes in the study of carbon fixation processes (in which TweCA and orthologue enzymes are involved) or drug candidates in the treatment of malaria.     

On the muscles of expression in the geladaTheropithecus gelada (Rüppell) (primates,Cercopithecidae)

Musculature innervated by the N. facialis inTheropithecus gelada (Rüppell) is patterned on broad lines in agreement with related genera of catarrhine monkeys, but presents some specializations and divergences in detail. Noteworthy is the extension to the labial margins superficially of the combined levator labii superioris and zygomaticus in the upper and the pars mandibularis of trachelo-platysma in the lower lip. A specialization of the medial fibres of levator labii superioris forms a sling-like structure within the upper lip and serves to implement the lip-flip gesture characteristic of the genus. Its antagonist is the orbicularis oris. Special features of all other facialis muscles are considered.Abbreviations ABD Anterior belly of digastricus - AE Arteria facialis - ALS Arteria labialis superior - ANL Arteria lateralis nasi - AP Auricularis posterior - APA Arteria auricularis posterior - AS Arteria auricularis superior - A.Se. Arteria septi nasi - A.Sy. Arteria symphysialis - BP Buccal pouch - LAO Depressor anguli oris - FTA Fronto-temporo-auricularis - GLI Glandulae labialis inferiores - GLS Glandulae labialis superioris - LAO Levator anguli oris - LG Artery to labial glands - LLAN Levator labii superioris alaeque nasi - LLS Levator labii superioris - M Masseter - MM Musculus mentalis - NP Notoplatysma - O Occipitalis - OO Orbicularis oris - O.Oc Orbicularis oculi - P Procerus - SH Sterno-hyoideus - TP Trachelo-platysma - VL Vena labialis communis - VP Venous plexus of dorsum nasi - ZM Zygomaticus minor - Zy Zygomaticus     

Organization of the lamina ganglionaris of the optic lobe of the butterfly Pararge aegeria (Linné) (Lepidoptera : Satyridae)

The present work reports on a neuroanatomical study of the butterfly Pararge aegeria (Lepidoptera : Satyridae) focusing on the lamina ganglionaris underlying two different regions of the retina of the compound eye: the dorsal rim area and the large dorsal region. No differences between both lamina regions, concerning the structure of the cartridges and the morphology of the identified neurons, could be detected. After passing the basement membrane, the visual cell axons are organized in retinotopic bundles (pseudocartridges), in which the axons of the 9 visual cells (V1 and 5, D2, 4, 6, 8, H3 and 7, B9) are arranged in the same way as in the retina. In the pseudocartridge there are no synaptic contacts. Before entering the lamina cartridge, the bundles rotate 90 °. The cartridges are joined by the fibres of 4 monopolar cells (L1, L2, L3 and L4), which could be identified and located inside the lamina cartridges in serial EM-sections. Golgi impregnations revealed the morphology of these fibres. Thus, the regional specialization of the retina (dorsal rim area and large dorsal region) does not seem to be reflected at the level of the first visual neuropil. Additionally, the cartridges of both lamina regions were investigated qualitatively for synaptic contacts among fibres. In addition to monadic chemical synapses and multiple contact synapses with presynaptic ribbons, cell contacts are also facilitated by invaginations and bridges. These cellular interactions and their functional implications are discussed.     

Phylogeny of the Alismatales (Monocotyledons) and the relationship of Acorus (Acorales?)

A phylogenetic analysis of the early branching lineages of the monocotyledons is performed using data from two plastid genes (rbcL and matK), five mitochondrial genes (atp1, ccmB, cob, mttB and nad5) and morphology. The complete matrix includes 93 terminals representing Acorus, the 14 families currently recognized within Alismatales, and numerous lineages of monocotyledons and other angiosperms. Total evidence analysis results in an almost completely resolved strict consensus tree, but all data partitions, genomic as well as morphological, are incongruent. The effects of RNA editing and potentially processed paralogous sequences are explored and discussed. Despite a decrease in incongruence length differences after exclusion of edited sites, the major data partitions remain significantly incongruent. The 14 families of Alismatales are all found to be monophyletic, but Acorus is found to be included in Alismatales rather than being the sister group to all other monocotyledons. The placement is strongly supported by the mitochondrial data, atp1 in particular, but it cannot be explained as an artifact caused by patterns of editing or by sampling of processed paralogues.     

Mosquitoes (Diptera: Culicidae) of the Lower Dyje River Basin (Podyjí) at the Czech-Austrian border

During 2009–2011, mosquitoes were captured in CDC miniature light traps using CO₂ (dry ice) at six sites in the Lower Dyje River Basin (Czech Republic). Other methods of capture — sweeping from vegetation and collection of larvae and pupae from ponds — were also used for more precise diagnostics. Thirty mosquito species of six genera were confirmed. A total of 415,218 females were captured. Most frequently found were the outbreak species Aedes vexans (56.52% of all mosquitoes collected) and Ae. sticticus (16.40%). Among other flood species, Ae. rossicus (5.17%), Ae. cantans and Ae. annulipes (2.44% of all females collected), and Ae. cinereus s. l. (1.11%) were especially abundant. Females of Ae. cataphylla were captured in spring (0.31%) and Ae. intrudens was numerous only at one site. Among the other species, Culex pipiens s. l. (6.61%) and Cx. modestus (8.87%) were abundant. Anopheles maculipennis s. l. (1.01%), An. claviger (0.43%), An. plumbeus (0.08%), An. hyrcanus (0.08%), Coquillettidia richiardii (0.52%) and Culiseta annulata (0.18%) were also detected. Sparsely occurring were Ae. excrucians, Ae. flavescens, Ae. caspius and Ae. geniculatus. Captured only very sporadically were Ae. communis, Ae. leucomelas, Ae. dorsalis, Ae. rusticus, Cx. martinii, Cx. territans, Cs. morsitans and Uranotaenia unguiculata.