Carbonic anhydrase inhibitors: synthesis of Schiff bases of hydroxybenzaldehydes with aromatic sulfonamides and their reactions with arylsulfonyl isocyanates

Reaction of o- or p-hydroxybenzaldehydes with sulfanilamide, homosulfanilamide and p-(2-aminoethyl)- benzene-sulfonamide afforded several new Schiff bases which were subsequently derivatized at the phenolic hydroxy moiety by reaction with arylsulfonylisocyanates. The new arylsulfonylcarbamates obtained in this way possessed interesting inhibitory properties against three carbonic anhydrase (CA) isozymes, hCA I, hCA II and bCA IV (h = human, b = bovine isozyme). All these new derivatives, the simple Schiff bases and the arylsulfonylcarbamates obtained as outlined above, were more inhibitory against all isozymes as compared to the corresponding parent sulfonamide from which they were obtained. Generally, the p-hydroxybenzaldehyde derivatives were more active than the corresponding ortho isomers. An interesting behavior was evidenced for some of the ortho-substituted arylsulfonylcarbamato-sulfonamides, which showed higher affinities for the isozyme hCA I, as compared to hCA II and bCA IV (generally hCA I is 10-1000 less sensitive to "normal" sulfonamide inhibitors, such as acetazolamide, methazolamide or dorzolamide, as compared to hCA II). This make the new derivatives attractive leads for designing isozyme I-specific inhibitors.     

Carbonic anhydrase inhibitors; phosphoryl-sulfonamides--a new class of high affinity inhibitors of isozymes I and II

A series of phosphorylated aromatic/heterocyclic sulfonamides with the general formula ArSO2NHPO3H2 have been prepared by condensing ArSO2NH2 with phosphorus pentachloride, followed by controlled hydrolysis in the presence of formic acid. The new derivatives generally act as stronger inhibitors of two carbonic anhydrase (CA) isozymes, CA I and CA II, as compared to the parent unsubstituted sulfonamides from which they were obtained. The inhibition mechanism by this new class of CA inhibitors, as well as structure activity correlations for the series of investigated derivatives, are also discussed.     

Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII

We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties.     

A Theoretical Model for the Association Probabilities of Saturated Phospholipids from Two-Component Bilayer Lipid Membranes

The non-random mixing of biomembrane components, especially saturated phospholipids, exhibits important consequences in molecular biology. Particularly, the distribution of lipids within natural and model membranes is strongly determined by the selective association processes. These processes of phospholipids take place due to the cooperative modes in multiparticle systems as well as the specific lipid-lipid interactions both in the hydrophobic core and in the region of the polar headgroups. We demonstrated that the investigation of the selective association processes of saturated phospholipids might contribute to the insight of the lipid domains appearance inside the bilayer membranes. The association probabilities of like-pairs and cross-pairs from a binary mixture of saturated phospholipids were tested for both parallel and anti-parallel alignments of the polar headgroups. The present model confirms the experimental evidence for saturated phospholipids to have a high tendency for association in parallel configuration of the electric dipole moments of the polar headgroups whether the cross-sectional area of the polar headgroup is in an usual range of 25-55 2. There are three major lipid domains in a binary mixture of saturated phospholipids: (i) lipid domains in non-mixed phase of the first mixture component, in parallel alignment of the polar headgroups; (ii) lipid domains in non-mixed phase of the second mixture component, in anti-parallel alignment of the polar headgroups; (iii) lipid domains in mixed phase. We think that the selective association processes of phospholipids are neither exclusively, nor only involved in promoting the lipid domains appearance through bilayer phospholipid membranes.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Sequential resonance assignment of medium-sized15 N/13C-labeled proteins with projected 4D triple resonance NMR experiments

We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of ¹⁵N/¹³C-labeled proteins. A 3D ^{/ /}(CO)NHN experiment is recorded either in conjunction with 3D HNN< > or with the newly presented 3D HNN scheme. The first combination yields sequential assignments based on the measurement of¹³ C chemical shifts and provides a complete ¹H, ¹³C and ¹⁵N resonance assignment of polypeptide backbone and CH_n moieties. When employing the second combination, ¹³C=O chemical shifts are not measured, but the sequential assignment relies on both ¹³C and¹ H chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13 °C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30 °C , demonstrates the efficiency of our approach for the assignment of medium-sized¹⁵ N/¹³C doubly labeled proteins.     

Vasoactive Intestinal Polypeptide Promotes Intestinal Barrier Homeostasis and Protection Against Colitis in Mice

Inflammatory bowel disease is a chronic gastrointestinal inflammatory disorder associated with changes in neuropeptide expression and function, including vasoactive intestinal peptide (VIP). VIP regulates intestinal vasomotor and secretomotor function and motility; however, VIP’s role in development and maintenance of colonic epithelial barrier homeostasis is unclear. Using VIP deficient (VIPKO) mice, we investigated VIP’s role in epithelial barrier homeostasis, and susceptibility to colitis. Colonic crypt morphology and epithelial barrier homeostasis were assessed in wildtype (WT) and VIPKO mice, at baseline. Colitic responses were evaluated following dinitrobenzene sulfonic acid (DNBS) or dextran-sodium sulfate (DSS) exposure. Mice were also treated with exogenous VIP. At baseline, VIPKO mice exhibited distorted colonic crypts, defects in epithelial cell proliferation and migration, increased apoptosis, and altered permeability. VIPKO mice also displayed reduced goblet cell numbers, and reduced expression of secreted goblet cell factors mucin 2 and trefoil factor 3. These changes were associated with reduced expression of caudal type homeobox 2 (Cdx2), a master regulator of intestinal function and homeostasis. DNBS and DSS-induced colitis were more severe in VIPKO than WT mice. VIP treatment rescued the phenotype, protecting VIPKO mice against DSS colitis, with results comparable to WT mice. In conclusion, VIP plays a crucial role in the development and maintenance of colonic epithelial barrier integrity under physiological conditions and promotes epithelial repair and homeostasis during colitis.     

No evidence of inhibition of horizontal gene transfer by CRISPR–Cas on evolutionary timescales

The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)–Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR–Cas maintenance and one of the causes of the patchy distribution of CRISPR–Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR–Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR–Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.     

Hemostasis During Urologic Surgery: Fibrin Sealant Compared With Absorbable Hemostat

In the United States, fibrin sealants have been used to achieve hemostasis for nearly two decades. Although their clinical utility was first demonstrated in cardiac surgery, their effectiveness and safety have since been demonstrated to extend to a wide array of procedures. Fibrin sealants typically contain two components—fibrinogen and thrombin—that are combined and delivered simultaneously to a target bleeding site in order to achieve hemostasis. However, many commercial formulations contain other additional components, such as antifibrinolytic agents, that have been associated with adverse outcomes. This subanalysis compares the safety and effectiveness of a fibrin sealant versus an absorbable hemostat for achieving hemostasis during urologic procedures with mild to moderate bleeding.Key words: Hemostasis, Hemostatics, Fibrin tissue adhesive, Urologic surgical procedures, Surgical techniqueIn the United States, fibrin sealants have been used to achieve hemostasis for nearly two decades. Although their clinical utility was first demonstrated in cardiac surgery,¹ their effectiveness and safety have since been demonstrated to extend to a wide array of procedures, including cardiovascular, gastrointestinal, pneumothoracic, neurologic, urologic, otolaryngologic, dental, and reconstructive surgeries.^2,3 Within the field of urology, fibrin sealants have been used to manage bleeding from renal trauma,⁴ as well as to facilitate hemostasis during renal surgeries, including partial nephrectomies.^5–7Fibrin sealants typically contain two components—fibrinogen and thrombin—that are combined and delivered simultaneously to a target bleeding site (TBS) in order to achieve hemostasis.³ Many commercial formulations contain other additional components, such as antifibrinolytic agents, that have been associated with adverse outcomes. For example, in an observational study (N = 4374), the antifibrinolytic aprotinin was associated with an increased risk of long-term mortality within 5 years following coronary artery bypass graft surgery.⁸ Furthermore, repeated exposure to aprotinin may lead to allergic or potentially fatal anaphylactic reactions.^9–11 For this reason, the US Food and Drug Administration (FDA) issued an alert in 2006 indicating that caution should be used when using aprotinin in patients with a history of previous exposure to the product.¹² Tranexamic acid, another antifibrinolytic present in some commercial fibrin sealants, has been associated with alterations in neural tissue growth and adherence.¹³ Because of the risk of cerebral neurologic toxicity, fibrin sealants containing tranexamic acid are contraindicated for use in neurosurgery or in surgical procedures during which contact with cerebrospinal fluid or dura mater may occur.¹⁴In a phase III, randomized, single-blind, parallel-group, multicenter study,¹⁵ the fibrin sealant CROSSEAL™ (Ethicon, Inc., Somerville, NJ) significantly reduced the time to hemostasis during liver resection surgery compared with conventional hemostatic techniques. EVICEL® Fibrin Sealant (Human) (Ethicon, Inc.), the successor of CROSSEAL, requires no antifibrinolytic additive and is therefore both aprotinin and tranexamic acid free, and achieves hemostasis using exclusively human components.¹⁶ The effectiveness and safety of this fibrin sealant for hemostasis in soft tissue during elective retroperitoneal or intra-abdominal surgery were compared with an absorbable hemostat (SURGICEL® Absorbable Hemostat; Ethicon, Inc.) in a randomized, active-controlled, multicenter study.¹⁷ This article describes a subanalysis of data from the largest patient subgroup from that study, and evaluates the effectiveness and safety of a fibrin sealant versus an absorbable hemostat for patients who underwent urologic surgical procedures.     

Highly Loaded Behavior of Kinesins Increases the Robustness of Transport Under High Resisting Loads

Kinesins are nano-sized biological motors which walk by repeating a mechanochemical cycle. A single kinesin molecule is able to transport its cargo about 1 μm in the absence of external loads. However, kinesins perform much longer range transport in cells by working collectively. This long range of transport by a team of kinesins is surprising because the motion of the cargo in cells can be hindered by other particles. To reveal how the kinesins are able to accomplish their tasks of transport in harsh intracellular circumstances, stochastic studies on the kinesin motion are performed by considering the binding and unbinding of kinesins to microtubules and their dependence on the force acting on kinesin molecules. The unbinding probabilities corresponding to each mechanochemical state of kinesin are modeled. The statistical characterization of the instants and locations of binding are captured by computing the probability of unbound kinesin being at given locations. It is predicted that a group of kinesins has a more efficient transport than a single kinesin from the perspective of velocity and run length. Particularly, when large loads are applied, the leading kinesin remains bound to the microtubule for long time which increases the chances of the other kinesins to bind to the microtubule. To predict effects of this behavior of the leading kinesin under large loads on the collective transport, the motion of the cargo is studied when the cargo confronts obstacles. The result suggests that the behavior of kinesins under large loads prevents the early termination of the transport which can be caused by the interference with the static or moving obstacles.