Isoform-specific requirement for GSK3α in sperm for male fertility.

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein kinase regulating key cellular functions. Its two isoforms, GSK3α and GSK3ß, are encoded by distinct genes. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3ß is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. Both GSK3 isoforms, present in sperm from several species including human, are suggested to play a role in epididymal initiation of sperm motility. Using genetic approaches, we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3 isoforms are expressed at high levels during the onset of spermatogenesis. Conditional knockout of GSK3α, but not GSK3ß, in developing testicular germ cells in mice results in male infertility. Mice lacking one allele each of GSK3α and GSK3ß are fertile. Despite overlapping expression and localization in differentiating spermatids, GSK3ß does not substitute for GSK3α. Loss of GSK3α impairs sperm hexokinase activity resulting in low ATP levels. Net adenine nucleotide levels in caudal sperm lacking GSK3α resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1γ2 with its protein interactors occurring during epididymal sperm maturation is impaired in sperm lacking GSK3α. The isoform-specific requirement for GSK3α is likely due to its specific binding partners in the sperm principal piece. Testis and sperm are unique in their specific requirement of GSK3α for normal function and male fertility.

The effect of methylated phosphatidylethanolamine derivatives on the ionization properties of signaling phosphatidic acid.

Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC) are the most abundant glycerophospholipids in eukaryotic membranes. The differences in the physicochemical properties of their headgroups have contrasting modulatory effects on their interaction with intracellular macromolecules. As such, their overall impact on membrane structure and function differs significantly. Enzymatic methylation of PE's amine headgroup produces two methylated derivatives namely monomethyl PE (MMPE) and dimethyl PE (DMPE) which have physicochemical properties that generally range between that of PE and PC. Additionally, their influence on membrane properties differs from both PE and PC. Although variations in headgroup methylation have been reported to affect signaling pathways, the direct influence that these differences exert on the ionization properties of signaling phospholipids have not been investigated. Here, we briefly review membrane function and structure that are mediated by the differences in headgroup methylation between PE, MMPE, DMPE and PC. In addition, using 31P MAS NMR, we investigate the effect of these four phospholipids on the ionization properties of the ubiquitous signaling anionic lipid phosphatidic acid (PA). Our results show that PA's ionization properties are differentially affected by changes in phospholipid headgroup methylation. This could have important implications for PA-protein binding and hence physiological functions in cells where signaling events lead to changes in abundance of methylated PE derivatives in the membrane.

Preparation and Characterization of Dual-Modified Cassava Starch-Based Biodegradable Foams for Sustainable Packaging Applications.

Starch and its derivatives have recently emerged as a sustainable and renewable alternative for petroleum-based expanded polystyrene (EPS) and expanded polypropylene (EPP) foam materials. In this study, biodegradable foam materials were prepared from cassava starch using a novel dual modification technique, combining microwave treatment and freeze-drying. The foam materials were prepared from starch solutions microwaved over different intervals. The starch-based foam materials were characterized using Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), X-ray diffraction (XRD), scanning electron microscopy (SEM), 13C nuclear magnetic resonance (13C-NMR) spectroscopy, and compression set test. Moreover, the water absorption capacities and density values of the foam materials were measured according to ASTM standards. The biodegradability test was carried out according to the aerobic compost environment test. The lowest water absorption capacities of 65.56% and 70.83% were exhibited for the cassava starch foam sample (MWB) prepared at a 20 s microwave treatment time and immersed in distilled water for 2 and 24 h, respectively. Furthermore, the lightweight cassava starch-based foam materials displayed density ranging from 124 to 245 kg/m3. The biodegradation test exhibited significant biodegradation of over 50% after 15 days for all the foam materials prepared. These results suggest that the dual-modified cassava starch-based biodegradable foams show potential in sustainable packaging applications by replacing petroleum-based materials.

Stability and ring-shift tautomerization of cyclic anhydrides of benzenehexasulfonic acid.

Upon heating in a dry atmosphere, benzenehexasulfonic acid forms three cyclic anhydrides. Mono- and dianhydride do not hydrolyze readily due their flatter structures compared to the hydrolysis products. The trianhydride appears more to be reactive toward hydrolysis. In solutions, the mono- and dianhydride undergo ring-shift tautomerization, which is in the latter case shifted toward the para isomer.

Ionization properties of phosphatidylinositol polyphosphates in mixed model membranes.

Phosphatidylinositol polyphosphate lipids (phosphoinositides) form only a minor pool of membrane phospholipids but are involved in many intracellular signaling processes, including membrane trafficking, cytoskeletal remodeling, and receptor signal transduction. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but also capable of forming hydrogen bonds. Many proteins have developed special binding domains that facilitate specific binding to particular phosphoinositides, while other proteins interact with phosphoinositides via nonspecific electrostatic interactions. Despite its importance, only limited information is available about the ionization properties of phosphoinositides. We have investigated the pH-dependent ionization behavior of all three naturally occurring phosphatidylinositol bisphosphates as well as of phosphatidylinositol 3,4,5-trisphosphate in mixed phosphoinositide/phosphatidylcholine vesicles using magic angle spinning (31)P NMR spectroscopy. For phosphatidylinositol 3,5-bisphosphate, where the two phosphomonoester groups are separated by a hydroxyl group at the 4-position, the pH-dependent chemical shift variation can be fitted with a Henderson-Hasselbalch-type formalism, yielding pK(a)(2) values of 6.96 +/- 0.04 and 6.58 +/- 0.04 for the 3- and 5-phosphates, respectively. In contrast, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] as well as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] show a biphasic pH-dependent ionization behavior that cannot be explained by a Henderson-Hasselbalch-type formalism. This biphasic behavior can be attributed to the sharing of the last remaining proton between the vicinal phosphomonoester groups. At pH 7.0, the overall charge (including the phosphodiester group charge) is found to be -3.96 +/- 0.10 for PI(3,4)P(2) and -3.99 +/- 0.10 for PI(4,5)P(2). While for PI(3,5)P(2) and PI(4,5)P(2) the charges of the individual phosphate groups in the molecule differ, they are equal for PI(3,4)P(2). Differences in the charges of the phosphomonoester groups can be rationalized on the basis of the ability of the respective phosphomonoester group to form intramolecular hydrogen bonds with adjacent hydroxyl groups. Phosphatidylinositol 3,4,5-trisphosphate shows an extraordinary complex ionization behavior. While at pH 4 the (31)P NMR peak of the 4-phosphate is found downfield from the other two phosphomonoester group peaks, an increase in pH leads to a crossover of the 4-phosphate, which positions this peak eventually upfield from the other two peaks. As a result, the 4-phosphate group shows a significantly lower charge at pH values between 7 and 9.5 than the other two phosphomonoester groups. The charge of the respective phosphomonoester group in PI(3,4,5)P(3) is lower than the corresponding charge of the phosphatidylinositol bisphosphate phosphomonoester groups, leading to an overall charge of PI(3,4,5)P(3) of -5.05 +/- 0.15 at pH 7.0. The charge of all investigated phosphoinositides at pH 7.0 is equal or higher than the corresponding charge of soluble inositol polyphosphate headgroup analogues, which is the opposite of what is expected on the basis of simple electrostatic considerations. This higher than expected headgroup charge can be rationalized with mutual intermolecular hydrogen bond formation. Measurements using different concentrations of PI(4,5)P(2) in the lipid vesicles (1, 5, and 20 mol %) did not reveal any significant concentration-dependent shift of the two phosphomonoester peaks, suggesting that PI(4,5)P(2) is clustered even at 1 mol %.

Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase.

Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS (nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.

Coordination-driven self-assembly of a Pt(iv) prodrug-conjugated supramolecular hexagon.

This article presents a new strategy to engage coordination-driven self-assembly for platinum drug delivery. The self-assembled supramolecular hexagon is conjugated with three equivalents of Pt(iv) prodrugs and displays a superior therapeutic index compared to cisplatin against a panel of human cancer cell lines.

Multinuclear nuclear magnetic resonance spectroscopic and high-performance liquid chromatographic characterization of silica, grafted with specifically deuterated 4-((propylamino)methyl)benzoic acid.

Specifically deuterated 4-((propylamino)methyl)benzoic acid-grafted silica (PAMBA-silica) was prepared by benzylation of propylamino-grafted silica (PA-silica) by either in situ reduction by sodium cyanoborodeuteride (NaCNBD3) of the Schiff base, formed by the reaction between PA-silica and 4-formylbenzoic acid, or by NaCNBD3 reduction of the isolated Schiff base. The PAMBA-silicas, which contain amine and carboxylic acid functionalities, were characterized by elemental analysis, (13)C, (29)Si, and (2)H solid state NMR, and HPLC. Solid state (13)C NMR revealed that PAMBA-silica prepared by the in situ method consists of di-benzylated, mono-benzylated, and unreacted amino-groups while PAMBA-silica prepared by the two-step synthesis consists of only mono-benzylated and unreacted amino-groups. (29)Si solid-state NMR spectra indicated that no significant loss of propylamine groups had occurred during benzylation. Nearly ideal uniaxial rigid-limit (2)H NMR spectra of grafted 4-PAMBA ligands indicates that they form a rigid structure, which provides effective electrostatic screening of inner positive charges when the ligands are in zwitterionic form. HPLC columns packed with PAMBA-silica and PA-silica were evaluated for ionic solutes at different pH of the mobile phase. Retention times increased for cations and decreased for anions at increasing pH. These trends show that PAMBA-silicas act as cation and anion exchangers at high and low pH, respectively. The pKa values of grafted carboxylic acid, determined from HPLC of weakly retaining solutes, are close to pKa of the solution PAMBA.

Tetrahydrobiopterin redox cycling in nitric oxide synthase: evidence supports a through-heme electron delivery.

The nitric oxide synthases (NOS) catalyze a two-step oxidation of l-arginine (Arg) to generate NO. In the first step, O2 activation involves one electron being provided to the heme by an enzyme-bound 6R-tetrahydro-l-biopterin cofactor (H4 B), and the H4 B radical must be reduced back to H4 B in order for NOS to continue catalysis. Although an NADPH-derived electron is used to reduce the H4 B radical, how this occurs is unknown. We hypothesized that the NOS flavoprotein domain might reduce the H4 B radical by utilizing the NOS heme porphyrin as a conduit to deliver the electron. This model predicts that factors influencing NOS heme reduction should also influence the extent and rate of H4 B radical reduction in kind. To test this, we utilized single catalytic turnover and stop-freeze methods, along with electron paramagnetic resonance spectroscopy, to measure the rate and extent of reduction of the 5-methyl-H4 B radical formed in neuronal NOS (nNOS) during Arg hydroxylation. We used several nNOS variants that supported either a slower or faster than normal rate of ferric heme reduction. We found that the rates and extents of nNOS heme reduction correlated well with the rates and extents of 5-methyl-H4 B radical reduction among the various nNOS enzymes. This supports a model where the heme porphyrin transfers an electron from the NOS flavoprotein to the H4 B radical formed during catalysis, revealing that the heme plays a dual role in catalyzing O2 activation or electron transfer at distinct points in the reaction cycle.