The N-terminal part of Binder of SPerm 5 (BSP5), which promotes sperm capacitation in bovine species is intrinsically disordered

Bovine BSP5 belongs to the Binder of SPerm (BSP) family. BSP5 plays a role in the bovine sperm capacitation by promoting cholesterol and phospholipid efflux. The variable N-terminal part in the BSP proteins is the uncharacterized region with no known function. Full-length, N-terminal part, and individual fibronectin type II domains of bovine BSP5 were cloned, expressed and purified from Escherichia coli. His-S tagged N-terminal part showed large variation in migration on SDS-PAGE in comparison to other constructs. Using mass spectrometry it was demonstrated that the His-S-N-terminal part has the expected molecular mass (13 kDa). The recombinant N-terminal part was sensitive to E. coli endogenous proteases during purification. Denaturing purification involving boiling lysis of cells was carried out, as the protein was thermostable. The His-S-N-terminal part lacked structure as determined by CD analysis. Bioinformatics analyses confirmed that the N-terminal part of bovine BSP5 is intrinsically disordered. In addition, bioinformatics analysis indicated that rabbit BSP and multiple forms of BSP proteins of bovine and equine species possess partially or completely disordered N-terminus. The conservation of disorder at the N-terminus in BSP members belonging to different species suggests a role in biological process such as sperm capacitation and/or sperm-egg interactions.     

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

An electron spin resonance study of the novel radical cation produced during the horseradish peroxidase-catalyzed oxidation of tetramethylhydrazine

Thrombin stimulation of [³²P]-prelabeled platelets induces a rapid decrease of the radioactivity from phosphatidylinositol-4,5-bisphosphate. No significant change is observed in phosphatidylinositol-4-monophosphate. The initial, thrombin-induced decrease of phosphatidylinositol-4,5-bisphosphate is not inhibited by cytochalasin D or by compounds that interfere with the mobilization of Ca²⁺ such as 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the calmodulin-antagonist, trifluoperazine, prostacyclin and cyclic AMP. Our information indicates that the rapid loss of phosphatidylinositol-4,5-bisphosphate is linked to receptor activation and insensitive to Ca²⁺-mobilization.     

Stability of Benzocaine Formulated in Commercial Oral Disintegrating Tablet Platforms

Pharmaceutical excipients contain reactive groups and impurities due to manufacturing processes that can cause decomposition of active drug compounds. The aim of this investigation was to determine if commercially available oral disintegrating tablet (ODT) platforms induce active pharmaceutical ingredient (API) degradation. Benzocaine was selected as the model API due to known degradation through ester and primary amino groups. Benzocaine was either compressed at a constant pressure, 20 kN, or at pressure necessary to produce a set hardness, i.e., where a series of tablets were produced at different compression forces until an average hardness of approximately 100 N was achieved. Tablets were then stored for 6 months under International Conference on Harmonization recommended conditions, 25°C and 60% relative humidity (RH), or under accelerated conditions, 40°C and 75% RH. Benzocaine degradation was monitored by liquid chromatography–mass spectrometry. Regardless of the ODT platform, no degradation of benzocaine was observed in tablets that were kept for 6 months at 25°C and 60% RH. After storage for 30 days under accelerated conditions, benzocaine degradation was observed in a single platform. Qualitative differences in ODT platform behavior were observed in physical appearance of the tablets after storage under different temperature and humidity conditions.     

Factors Controlling the Redox Potential of ZnCe6 in an Engineered Bacterioferritin Photochemical ‘Reaction Centre’

Photosystem II (PSII) of photosynthesis has the unique ability to photochemically oxidize water. Recently an engineered bacterioferritin photochemical ‘reaction centre’ (BFR-RC) using a zinc chlorin pigment (ZnCe₆) in place of its native heme has been shown to photo-oxidize bound manganese ions through a tyrosine residue, thus mimicking two of the key reactions on the electron donor side of PSII. To understand the mechanism of tyrosine oxidation in BFR-RCs, and explore the possibility of water oxidation in such a system we have built an atomic-level model of the BFR-RC using ONIOM methodology. We studied the influence of axial ligands and carboxyl groups on the oxidation potential of ZnCe₆ using DFT theory, and finally calculated the shift of the redox potential of ZnCe₆ in the BFR-RC protein using the multi-conformational molecular mechanics–Poisson-Boltzmann approach. According to our calculations, the redox potential for the first oxidation of ZnCe₆ in the BRF-RC protein is only 0.57 V, too low to oxidize tyrosine. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe₆ di-cation. In order to increase the efficiency of tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe₆ would have to attain a value in excess of 0.8 V. We discuss the possibilities for modifying the BFR-RC to achieve this goal.     

Barrier potentials,molecular structure,force filed calculations and quantum chemical studies of some bipyridine di-carboxylic acids using the experimental and theoretical using (DFT,IVP) approach

ABSTRACT

FT-IR and FT-Raman spectra of 2,2′-bipyridine-3,3′-dicarboxylic acid (B3DA), 2,2′-bipyridine-4,4′-dicarboxylic acid (B4DA) and 2,2′-bipyridine-5,5′-dicarboxylic acid (B5DA) were recorded and analysed. The quantum chemical calculations of the title compounds begin with barrier potentials at different rotation angles around the C–C′ and C–Cα bonds in order to arrive conformation of lowest energy using DFT employing B3LYP functional with 6-311++G(d,p) basis set. This confirmation was further optimised to get the global minimum geometry. The vibrational frequencies along with IR, Raman intensities were computed, the rms error between observed and calculated frequencies were 11.2 cm^?1, 10.2 cm^?1 and 12.2 cm^?1 for B3DA, B4DA, and B5DA. An 87-element modified valence force field is derived by solving the inverse vibrational problem using Wilson’s GF matrix method. This force field is refined using 163 observed fundamentals employing in overlay least-squares technique. The average error between computed and experimental frequencies was found as 12.85 cm^?1 using potential energy distribution (PED) and eigenvectors. By using the gauge-independent atomic orbital (GIAO) method calculate the ¹H and ¹³C NMR chemical shifts of the molecules and compared with experimental results. The first-order hyperpolarisability, HOMO and LUMO energies, molecular electrostatic potential (MESP) and natural orbital analysis (NBO) of titled compounds were evaluated using DFT.     

Synthesis,angiopreventive activity,and in vivo tumor inhibition of novel benzophenone–benzimidazole analogs

Aim

The development of anticancer drugs with specific targets is of prime importance in modern biology. This study investigates the angiopreventive and in vivo tumor inhibition activities of novel synthetic benzophenone–benzimidazole analogs.

Main methods

The multistep synthesis of novel benzophenone–benzimidazole analogs (8a–n) allowing substitution with methoxy, methyl and halogen groups at different positions on the identical chemical backbone and the variations in the number of substituents were synthesized and characterized. The newly synthesized compounds were further evaluated for cytotoxic and antiproliferative effects against Ehrlich ascites carcinoma (EAC) cells. The potent lead compounds were further assessed for antiangiogenic effects in a CAM model and a tumor-induced vasculature in vivo model. The effect of angioprevention on tumor growth was verified in a mouse model.

Key findings

The cytotoxicity studies revealed that compounds 8f and 8n are strongly cytotoxic. Analyzing the structure–activity relationship, we found that an increase in the number of methyl groups in addition to methoxy substitution at the para position of the benzoyl ring in compound 8n resulted in higher potency compared to 8f. Furthermore, neovessel formation in in vivo systems, such as the chorioallantoic membrane (CAM) and tumor-induced mice peritoneum models, was significantly suppressed and reflected the tumor inhibition observed in mice.

Significance

These results suggest the potential clinical application of compound 8n as an antiangiogenic drug for cancer therapy.