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-tetrafluorophenylsulfonyl- 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.     

Strand displacement of double-stranded DNA by triplex-forming antiparallel purine-hairpins

We characterize the binding affinity and the thermodynamics of hybridization of triplex-forming antiparallel purine-hairpins composed of two antiparallel purine domains linked by a loop directed toward single-stranded and double-stranded DNA (ssDNA, dsDNA). Gel retardation assays and melting experiments reveal that a 13-mer purine-hairpin binds specifically and with a K ( d ) of 8 x 10(8) M to polypyrimidine ssDNA to form a triple helical structure. Remarkably, we show that purine-hairpins also bind polypurine/polypyrimidine stretches included in a dsDNA of several hundred bp in length. Binding of purine-hairpins to dsDNA occurs by triplex formation with the polypyrimidine strand, causing displacement of the polypurine strand. Because triplex formation is restricted to polypurine/polypyrimidine stretches of dsDNA, we studied the triplex formation between purine-hairpins and polypyrimidine targets containing purine interruptions. We found that an 11-mer purine-hairpin with an adenine opposite to a guanine interruption in the polypyrimidine track binds to ssDNA and dsDNA, allowing expansion of the possible target sites and increase in the length of purine-hairpins. Thus, when using a 20-mer purine-hairpin targeting an interruption-containing polypyrimidine target, the binding affinity is increased compared to its 13-mer antiparallel purine-hairpin counterpart. Surprisingly, this increase is much more pronounced than that observed for a tail-clamp purine-hairpin extended up to 20 nt in the Watson-Crick domain only. Thus, triplexforming antiparallel purine-hairpins can be a potentially useful strategy for both single-strand and double-strand nucleic acid recognition.     

A comparison of adrenal gland function in lactating dairy cows with or without ovarian follicular cysts

Two experiments were conducted to determine if adrenal secretion of steroids differed between cows that formed ovarian follicular cysts and normal cycling cows. In experiment 1, lactating Jersey and Holstein cows were diagnosed as having ovarian follicular cysts (follicle diameter >or=20 mm) by rectal palpation. Following diagnosis, ovaries were examined by transrectal ultrasonography three times weekly to detect subsequent ovulation (n=8) or new cyst formation (n=9). Venous blood samples were collected daily to quantify circulating concentrations of cortisol and progesterone. The average concentration of cortisol during the 10-day period prior to ovulation was not different from the concentration prior to the formation of a new cyst. In experiment 2, secretion of cortisol and progesterone was examined in cows with ovarian follicular cysts (n=4) and cyclic, control cows in the follicular phase of the estrous cycle (n=4). An adrenocorticotropic hormone (ACTH) challenge was administered to cystic cows 4-7 days after new cyst formation and to cyclic cows in the follicular phase of the cycle (36 h after induction of luteolysis). Jugular venous blood samples were collected at -60, -30, 0, +10, +20, +30, +60, +90, +120, +180, +240, +300 and +360 minutes relative to ACTH administration. A rapid increase in both cortisol and progesterone was observed immediately following administration of ACTH in each treatment group. Peak concentrations of both steroids were achieved within 60 minutes after administration of ACTH. Concentrations of cortisol and progesterone did not differ between cystic and cyclic cows. In summary, no differences in adrenal function were detected between normal cycling cows and cows with ovarian follicular cysts.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

Decorin evokes protracted internalization and degradation of the epidermal growth factor receptor via caveolar endocytosis

Decorin inhibits the epidermal growth factor receptor (EGFR) by down-regulating its tyrosine kinase activity, thereby blocking the growth of a variety of transformed cells and tumor xenografts. In this study we provide evidence that decorin directly binds to the EGFR causing its dimerization, internalization, and ultimately its degradation. Using various pharmacological agents to disrupt clathrin-dependent and -independent endocytosis, we demonstrate that decorin evokes a protracted internalization of the EGFR primarily via caveolar-mediated endocytosis. In contrast to EGF, decorin targets the EGFR to caveolae, but not to early or recycling endosomes. Ultimately, however, both EGF- and decorin-induced pathways converge into late endosomes/lysosomes for final degradation. Thus, we have discovered a novel biological mechanism for decorin that could explain its anti-proliferative and anti-oncogenic mode of action.     

Role of chloride ions in modulation of the interaction between von Willebrand factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.     

A malaria parasite-encoded vacuolar H(+)-ATPase is targeted to the host erythrocyte

The asexual development of malaria parasites inside the erythrocyte is accompanied by changes in the composition, structure, and function of the host cell membrane and cytoplasm. The parasite exports a membrane network into the host cytoplasm and several proteins that are inserted into the erythrocyte membrane, although none of these proteins has been shown to have enzymatic activity. We report here that a functional malaria parasite-encoded vacuolar (V)-H(+)-ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte. This localization was determined by separation of parasite and erythrocyte membranes and determination of enzyme marker activities and by immunofluorescence microscopy assays using antibodies against the B subunit of the malarial V-H(+)-ATPase and erythrocyte (spectrins) and parasite (merozoite surface protein 1) markers. Our results suggest that this pump has a role in the maintenance of the intracellular pH (pH(i)) of the infected erythrocyte. Our results also indicate that although the pH(i) maintained by the V-H(+)-ATPase is important for maximum uptake of small metabolites at equilibrium, it does not appear to affect transport across the erythrocyte membrane and is, therefore, not involved in the previously described phenomenon of increased permeability of infected erythrocytes that is sensitive to chloride channel inhibitors (new permeation pathway). This constitutes the first report of the presence of a functional enzyme of parasite origin in the plasma membrane of its host.