Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX (CA IX) and XII (CA XII) are involved in acidification of hypoxic tumors, a process correlated with poor prognosis and clinical outcome of patients harboring such tumors. This process may be reversed by inhibiting these enzymes with potent sulfonamide/sulfamate inhibitors. A series of such aromatic/heterocyclic sulfonamides incorporating 2,3,5,6-tetrafluorobenzoyl-, 2,3,5,6-tetrafluoro- phenylsulfonyl- and pentafluorophenylureido moieties has been investigated for its interaction with the catalytic domain of the human isozymes hCA IX and hCA XII. Some of these compounds showed excellent inhibitory properties against both isozymes IX and XII, with several subnanomolar inhibitors detected for the first time. These sulfonamides may constitute valuable candidates for the development of novel antitumor therapies based on the inhibition of such tumor-associated CA isozymes.     

Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.     

Applications of Hydrolytic and Decarboxylating Enzymes in Biotransformations

Peptides, and oligosaccharides and glycosides, can be synthesised by making use of the ‘reverse hydrolytic activity’ of proteases and glycosidases respectively. In applying these enzymes to the practical synthesis of these classes of compound, several factors need to be considered, namely the need to shift the rate-determining step through the use of activated substrates, the need to minimise competing hydrolysis of these and the need to minimise hydrolysis of the products. In spite of these problems, the enzymatic methods have many attractive features, not least amongst which is the absolute control of stereochemistry in acyl transfer and glycosyl transfer respectively.     

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis,but enhances DNA polymerase ζ – dependent spontaneous mutagenesis

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ.     

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

Diel vertical migration ofEudiaptomus gracilis during a short summer period

Several aspects of a diel vertical migration (DVM) of adultEudiaptomus gracilis in Lake Maarsseveen (The Netherlands) are described. The period of DVM lasted from the end of May until the middle of August. On May 21, 1989, the population was found divided into a deep dwelling part and a part in the upper five meter. Large shoals of juvenile perch were observed in the open water for the first time. On June 7, the whole population was down below 10 m and concentrated in a zone of high chlorphyll-a concentrations. One week later, a regular DVM was performed. The amplitude of this migration gradually decreased towards the end of the migration period. The ascent in the evening and the descent in the morning took place after sunset and before sunrise, respectively. The movements coincided with high relative changes in light intensity. Population size increased rapidly during the period of DVM but decreased again before the end of this period.     

The lifetime of molecular (Davydov) solitons

The lifetime of Davydov solitons in a one-dimensional system is studied theoretically. The process of thermalization and the properties of solitons at finite temperature are investigated and the processes of soliton creation and disintegration are discussed.     

Colonization and extinction of land planarians (Platyhelminthes,Tricladida) in a Brazilian Atlantic Forest regrowth remnant

Long-term assessments of species assemblages are valuable tools for detecting species ecological preferences and their dispersal tracks, as well as for assessing the possible effects of alien species on native communities. Here we report a 50-year-long study on population dynamics of the four species of land flatworms (Platyhelminthes, Tricladida, Terricola) that have colonized or become extinct in a 70-year-old Atlantic Forest regrowth remnant through the period 1955–2006. On the one hand, the two initially most abundant species, which are native to the study site, Notogynaphallia ernesti and Geoplana multicolor have declined over decades and at present do not exist in the forest remnant. The extinction of these species is most likely related with their preference for open vegetation areas, which presently do not exist in the forest remnant. On the other hand, the neotropical Geoplaninae 1 and the exotic Endeavouria septemlineata were detected in the forest only very recently. The long-term study allowed us to conclude that Geoplaninae 1 was introduced into the study area, although it is only known from the study site. Endeavouria septemlineata, an active predator of the exotic giant African snail, is originally known from Hawaii. This land flatworm species was observed repeatedly in Brazilian anthropogenic areas, and this is the first report of the species in relatively well preserved native forest, which may be evidence of an ongoing adaptive process. Monitoring of its geographic spread and its ecological role would be a good practice for preventing potential damaging effects, since it also feeds on native mollusk fauna, as we observed in lab conditions. Júlio Pedroni: Granted by CNPQ–Brazil.     

Tracking wind‐dispersed seeds using 15N‐isotope enrichment

Seed dispersal influences a wide range of ecological processes. However, measuring dispersal patterns, particularly long‐distance dispersal, has been a difficult task. Marking bird‐dispersed seeds with stable ¹⁵N isotopes has been shown to be a user‐friendly method to trace seed dispersal. In this study, we determined whether ¹⁵N urea solution could be used to enrich seeds of two common wind‐dispersed plants, Eupatorium glaucescens (Asteraceae) and Sericocarpus tortifolius (Asteraceae). We further tested if the water type (distilled versus tap) in ¹⁵N urea solutions influences the level and variability of enrichment of plant seeds, and if increasing spraying frequency per se increases enrichment. Because droughts may lower seed set or kill plants, we wanted to investigate if the additional use of an externally applied anti‐transpirant affects the intake of externally applied ¹⁵N into seeds. The results demonstrate that ¹⁵N enrichment of seeds can facilitate dispersal experiments with wind‐dispersed plants. The use of distilled water in ¹⁵N urea solutions did not increase ¹⁵N enrichment compared to tap water. Further, enrichment was more efficient at lower spray frequencies. Both the use of tap water and low frequencies could lower time, effort and project costs. The results suggest that species can be protected from drought using an anti‐transpirant without decreasing the incorporation of ¹⁵N into seeds.