Improved solubility of β-cyclodextrin inclusion complexes by using liquid ammonia as a solvent and the possibility of asymmetric reduction

Dramatic improvement in the poor solubility of β-cyclodextrin (β-CD) and its inclusion complexes in water was achieved by using liquid ammonia (liq. NH₃) instead of water as the solvent. Asymmetric NaBH₄ reduction of the carbonyl groups of the inclusion complexes in liq. NH₃ was examined in a homogeneous condition to give the corresponding alcohols with moderate chirality.     

Nutrient resorption patterns of plant functional groups in a tropical savanna: variation and functional significance

Green and senesced leaf nitrogen (N) and phosphorus (P) concentrations of different plant functional groups in savanna communities of Kruger National Park, South Africa were analyzed to determine if nutrient resorption was regulated by plant nutritional status and foliar N:P ratios. The N and P concentrations in green leaves and the N concentrations in senesced leaves differed significantly between the dominant plant functional groups in these savannas: fine-leaved trees, broad-leaved trees and grasses. However, all three functional groups reduced P to comparable and very low levels in senesced leaves, suggesting that P was tightly conserved in this tropical semi-arid savanna ecosystem. Across all functional groups, there was evidence for nutritional control of resorption in this system, with both N and P resorption efficiencies decreasing as green leaf nutrient concentrations increased. However, specific patterns of resorption and the functional relationships between nutrient concentrations in green and senesced leaves varied by nutrient and plant functional group. Functional relationships between N concentrations in green and senesced leaves were indistinguishable between the dominant groups, suggesting that variation in N resorption efficiency was largely the result of inter-life form differences in green leaf N concentrations. In contrast, observed differences in P resorption efficiencies between life forms appear to be the result of both differences in green leaf P concentrations as well as inherent differences between life forms in the fraction of green leaf P resorbed from senescing leaves. Our results indicate that foliar N:P ratios are poor predictors of resorption efficiency in this ecosystem, in contrast to N and P resorption proficiencies, which are more responsive to foliar N:P ratios.     

Synthesis of novel tricyclic oxazolidinones by a tandem SN2 and SNAr reaction: SAR studies on conformationally constrained analogues of Linezolid

A series of conformationally constrained analogues of Linezolid were synthesised by employing a tandem SN(2) and SNAr reaction as the key step and tested for antibacterial activity. While the hexahydroazolo-quinoxaline compounds were inactive, the tetrahydroazolo-benzothiazine compounds exhibited interesting antibacterial activity. The introduction of fluorine in the aromatic ring further made the compounds more potent in acetamide compounds resulting in an interesting analogue 32. However, the introduction of fluorine (analogue 34) on the already potent non-fluorine thiocarbamate 21 did not have any influence on the activity.     

Inhibition of NO3- and NO2- reduction by microbial Fe(III) reduction: evidence of a reaction between NO2- and cell surface-bound Fe2+

A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO(3)(-) and NO(2)(-) (NO(x)(-)) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe(2+) and NO(2)(-). This coating could then inhibit reduction of NO(x)(-) by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of approximately 3 mM NO(3)(-) reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe(2+) and NO(2)(-), demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe(2+) onto Paracoccus denitrificans inhibited NO(x)(-) reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO(2)(-) was chemically reduced to N(2)O by goethite or cell-sorbed Fe(2+), but not at appreciable rates by aqueous Fe(2+). Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe(2+) and NO(2)(-). The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.     

Splicing factor SF4 is dispensable for the assembly of a functional splicing complex and participates in the subsequent steps of the splicing reaction.

The splicing of nuclear messenger RNA precursors (pre-mRNA) can be reconstituted in vitro with factors partially purified from HeLa cell nuclear extracts. Splicing complexes are assembled in the presence of the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6 and the protein factors SF1, SF2, SF3 and U2AF. However, the complexes thus formed are inactive, i.e. they only contain unprocessed pre-mRNA. The intermediates and products of the splicing reaction are generated after addition of SF4. This splicing factor is a heat-labile protein which requires sulfhydryl groups for its activity. SF4 appears to participate, directly or indirectly, in the conversion of a functional but inactive splicing complex to the active spliceosome.     

Radiolytic reduction of protein and nonprotein disulfides in the presence of formate: a chain reaction

We have reported recently that the disulfide groups in bovine serum albumin can be reduced by a radiolytic chain reaction which occurs in deoxygenated solutions containing formate ions. This reaction, which involves the reduction of disulfide groups by hydrated electrons and carbon dioxide radical anions, has now been studied in greater detail and compared with an analogous reaction in small, disulfide containing molecules over a range of pH values and substrate concentrations. A two-step reaction is proposed to account for the reduction of disulfides in reactions which can have chain lengths of 20 or more. Thiols produced by the disulfide reduction are stable to the conditions of the reaction. For example, a biological assay showed that the integrity of glutathione was maintained even at radiation doses much larger than those required to achieve complete reduction of glutathione disulfide. It was found that the extent of disulfide reduction could easily be controlled by varying the radiation dose delivered to the solutions. Radiolytic reduction is a very useful way of reducing protein and low molecular weight disulfides without the use of excess quantities of reagents such as dithiothreitol. In many cases, the reaction solutions could be used directly for subsequent reactions and this may be of considerable value in modifying the structure of hormones, enzymes, membrane receptors, and other disulfide containing proteins. If ammonium formate is used, freeze drying is an effective way to remove the formate salt, should this be required.     

Peroxynitrite induces long-lived tyrosyl radical(s) in oxyhemoglobin of red blood cells through a reaction involving CO2 and a ferryl species

Peroxynitrite-mediated oxidative chemistry is currently the subject of intense investigation owing to the toxic side effects associated with nitric oxide overproduction. Using direct electron spin resonance spectroscopy (ESR) at 37 degrees C, we observed that in human erythrocytes peroxynitrite induced a long-lived singlet signal at g = 2.004 arising from hemoglobin. This signal was detectable in oxygenated red blood cells and in purified oxyhemoglobin but significantly decreased after deoxygenation. The formation of the g = 2.004 radical required the presence of CO2 and pH values higher than the pKa of peroxynitrous acid (pKa = 6.8), indicating the involvement of a secondary oxidant formed in the interaction of ONOO- with CO2. The g = 2.004 radical yield leveled off at a 1:1 ratio between peroxynitrite and oxyhemoglobin, while CO-hemoglobin formed less radical and methemoglobin did not form the radical at all. These results suggest that the actual oxidant is or is derived from the ONOOCO2- adduct interacting with oxygenated FeII-heme. Spin trapping with 2-methyl-2-nitrosopropane (MNP) of the g = 2.004 radical and subsequent proteolytic digestion of the MNP/hemoglobin adduct revealed the trapping of a tyrosyl-centered radical(s). A similar long-lived unresolved g = 2.004 singlet signal is a common feature of methemoglobin/H2O2 and metmyoglobin/H2O2 systems. We show by spin trapping that these g = 2.004 signals generated by H2O2 also indicated trapping of radicals centered on tyrosine residues. Analysis of visible spectra of hemoglobin treated with peroxynitrite revealed that, in the presence of CO2, oxyhemoglobin was oxidized to a ferryl species, which rapidly decayed to lower iron oxidation states. The g = 2.004 radical may be an intermediate formed during ferrylhemoglobin decay. Our results describe a new pathway of peroxynitrite-dependent hemoglobin oxidation of dominating importance in CO2-containing biological systems and identify the g = 2.004 radical(s) formed in the process as tyrosyl radical(s).     

Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP-VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I-K44A impaired GFP-VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I-K44A generated vesicles. A fusion protein containing the VAChT C-terminal tail precipitated the AP-2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C-terminal tail of VAChT interacts with the micro subunit of AP-2 in a di-leucine (L485A/L486A) dependent fashion. These observations suggest that the di-leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.     

Heme-bound nitroxyl, hydroxylamine, and ammonia ligands as intermediates in the reaction cycle of cytochrome c nitrite reductase: a theoretical study

In this article, we consider, in detail, the second half-cycle of the six-electron nitrite reduction mechanism catalyzed by cytochrome c nitrite reductase. In total, three electrons and four protons must be provided to reach the final product, ammonia, starting from the HNO intermediate. According to our results, the first event in this half-cycle is the reduction of the HNO intermediate, which is accomplished by two PCET reactions. Two isomeric radical intermediates, HNOH^? and H₂NO^?, are formed. Both intermediates are readily transformed into hydroxylamine, most likely through intramolecular proton transfer from either Arg₁₁₄ or His₂₇₇. An extra proton must enter the active site of the enzyme to initiate heterolytic cleavage of the N–O bond. As a result of N–O bond cleavage, the H₂N⁺ intermediate is formed. The latter readily picks up an electron, forming H₂N^+?, which in turn reacts with Tyr₂₁₈. Interestingly, evidence for Tyr₂₁₈ activity was provided by the mutational studies of Lukat (Biochemistry 47:2080, 2008), but this has never been observed in the initial stages of the overall reduction process. According to our results, an intramolecular reaction with Tyr₂₁₈ in the final step of the nitrite reduction process leads directly to the final product, ammonia. Dissociation of the final product proceeds concomitantly with a change in spin state, which was also observed in the resonance Raman investigations of Martins et al. (J Phys Chem B 114:5563, 2010).     

Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction

The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (approximately 11 micrograms/10(6) cells/48 h) in rodent cells using gene-linked co-amplification. The soluble forms of IL-2R were shown to retain IL-2 affinity shown by cell-surface IL-2R (Kd approximately 18 nM) and were purified to homogeneity using IL-2 affinity chromatography. Purified, recombinant IL-2R and biotinylated IL-2 were used to establish a solid-phase receptor binding assay. Binding of IL-2-biotin was demonstrated to be dose-dependent at concentrations ranging from 10 to 1000 ng/ml, and the specificity of receptor-ligand binding was demonstrated by competition with non-biotinylated IL-2 and with anti-receptor antibodies known to block IL-2 binding in vivo. This immunosorbent receptor assay offers a simple and rapid method for studying the binding of IL-2 to human IL-2R.     

Suppression of insulin release by galanin and somatostatin is mediated by a G-protein. An effect involving repolarization and reduction in cytoplasmic free Ca2+ concentration

The effects of galanin and somatostatin on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration [( Ca2+]i) were investigated using beta-cells isolated from obese hyperglycemic mice. Whereas insulin release was measured in a column perifusion system, membrane potential and [Ca2+]i were measured with the fluorescent indicators bisoxonol (bis-(1,3-diethylthiobarbiturate)trimethineoxonol) and quin 2, in cell suspensions in a cuvette. Galanin (16 nM) and somatostatin (400 nM) suppressed glucose-stimulated insulin release in parallel to promoting repolarization and a reduction in [Ca2+]i. The reduction in [Ca2+]i comprised an initial nadir followed by a slow rise and the establishment of a new steady state level. The slow rise in [Ca2+]i was abolished by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. Both peptides suppressed insulin release even when [Ca2+]i was raised by 25 mM K+. Under these conditions the inhibition of insulin release was partly reversed by an increase in the glucose concentration. Addition of 5 mM Ca2+ to a cell suspension, incubated in the presence of 20 mM glucose and either galanin, somatostatin, or the alpha 2-adrenergic agonist clonidine (10 nM), induced oscillations in [Ca2+]i, this effect disappearing subsequent to the addition of D-600. The effects of galanin, somatostatin, and clonidine on [Ca2+]i were abolished in beta-cells treated with pertussis toxin. In accordance with measurements of [Ca2+]i, treatment with pertussis toxin reversed the inhibitory effect of galanin on insulin release. The inhibitory action of galanin and somatostatin on insulin release is probably accounted for by not only a repolarization-induced reduction in [Ca2+]i and a decreased sensitivity of the secretory machinery to Ca2+, but also by a direct interaction with the exocytotic process. It is proposed that these effects are mediated by a pertussis toxin-sensitive GTP-binding protein.     

Extracellular Mg2+ induces a loss of microvilli, membrane retrieval, and the subsequent cortical reaction, in the oocyte of the prawn Palaemon serratus

Surface changes induced by sea water were analyzed in the ovulated oocyte of the prawn Palaemon serratus. They depended on the presence of external Mg2+ but not on external Ca2+ alone. Increasing external Mg2+ from 0 mM to 30 mM stimulated first a progressive disappearance of preexisting microvilli, which was over within 30 min of incubation. This is correlated with membrane removal via internalization of coated vesicles, ascertained by observations of endocytosis of an extracellular fluid-phase marker and by measurement of a diminution in membrane capacitance (Cm). Thirty-five minutes after sea water contact, the prawn oocyte underwent a cortical reaction independent of fertilization. It consists in a heavy exocytosis of ring-shaped elements, leading to the deposition of a thick capsule, and requiring a threshold Mg2+ concentration of greater than or equal to 10 mM and at least a 3-min incubation with Mg2+. Concurrently, the values of the membrane capacitance (Cm) and conductance (Gm) increased about 2 and 10 times their initial values, respectively. The calcium ionophore ionomycin, added to Mg(2+)-free artificial sea water, stimulated the cortical reaction with requirement of external Ca2+. Other divalent cations (Mn2+, Zn2+, Co2+, Ni2+, Cd2+) instead of Mg2+, induced the cortical reaction, but Ba2+, Sr2+, and La3+ did not. When eggs are fertilized, the cortical reaction takes place in two steps, the first being a discrete exocytosis of a foamy material and the second always involving ring-shaped elements.     

Oxidation/reduction of methionine residues in CCK: a study by radioimmunoassay and isocratic reverse phase high pressure liquid chromatography

The study was undertaken to investigate the oxidation and reduction of cholecystokinin (CCK) both as pure standards and as endogenous porcine peptides. Furthermore an attempt was made to prevent oxidation of the endogenous porcine peptides in the extraction procedure. CCK-8 and CCK-33 standards were always oxidized in weak solutions, CCK-8 varying from 26% to 67% oxidized and CCK-33 from 18% to 70%. Similarly, tissue extracts of porcine brain and duodenum contained oxidized forms of the peptide. CCK standards were readily oxidized in the presence of hydrogen peroxide. Oxidized CCK-8 standard and CCK-8 in porcine brain was 90% reduced and oxidized CCK-33 standard and in duodenal extracts was reduced by 70% by a 40 hour incubation with 0.725 mol/l dithiothreitol at 37 degrees C. Extraction of CCK peptides in the presence of 65 mmol/l dithiothreitol resulted in almost complete prevention of oxidation with over 95% of the peptides being obtained in the reduced state. This additive is therefore recommended for all tissue quantitation studies.     

Proton uptake upon quinone reduction in bacterial reaction centers: IR signature and possible participation of a highly polarizable hydrogen bond network

The light-induced Q _A ^– /Q_A FTIR difference spectra of Rb. sphaeroides and Rp. viridis show very broad positive bands of small amplitude peaking around 2750 cm^–1. Upon ¹H/²H exchange these bands shift to about 2150 cm^–1. Similarly, the Q _B ^– /Q_B spectra exhibit broad continuum bands at 2600 and 2800 cm^–1 shifting to 2100 and 2200 cm^–1 in ²H₂O for Rb. sphaeroides and Rp. viridis, respectively. These continuum bands are tentatively interpreted in terms of highly polarizable hydrogen bonds in a large web of polar bonds involving cofactors, amino acid residues, and structured water molecules. As a working hypothesis, we propose that the protons participating in this web redistribute upon quinone reduction, increasing their concentration around the newly formed charged species, and leading to net proton uptake. Assuming that the precise localization of the mobile protons is dependent on the local electrostatic, this model can explain the apparent discrepancies between some results of FTIR experiments and of electrostatic calculations. Notably, it could help rationalize the observation that mobile protons tend to localize on Glu L212 upon Q_B reduction in Rb. sphaeroides, while for Q_B reduction in Rp. viridis and for Q_A reduction in both Rb. sphaeroides and Rp. viridis, proton uptake by a small number of carboxylic residues is not supported by the FTIR data.     

A three-step protocol for lead optimization: quick identification of key conformational features and functional groups in the SAR studies of non-ATP competitive MK2 (MAPKAPK2) inhibitors

A three-step protocol for SAR development was introduced and applied to the SAR studies of the MK2 inhibitor program. Following this protocol, key conformational features and functional groups for improving MK2 inhibitor activity were quickly identified. Through this effort, the initial gap observed between in vitro binding activity and cellular activity in the lead identification stage was very much reduced. Compound 28 was identified with single digit binding activity (IC(50)=8 nM) and good cellular activity (EC(50)=310 nM). This provides further evidence that non-ATP-competitive binding MK2 inhibitors are feasible by targeting the outside ATP pocket.     

Methionine-mediated repression in Saccharomyces cerevisiae: a pleiotropic regulatory system involving methionyl transfer ribonucleic acid and the product of gene eth2

Detailed study of methionine-mediated repression of enzymes involved in methionine biosynthesis in Saccharomyces cerevisiae led to classification of these enzymes into two distinct regulatory groups. Group I comprises four enzymes specifically involved in different parts of methionine biosynthesis, namely, homoserine-O-transacetylase, homocysteine synthetase, adenosine triphosphate sulfurylase, and sulfite reductase. Repressibility of these enzymes is greatly decreased in strains carrying a genetically impaired methionyl-transfer ribonucleic acid (tRNA) synthetase (mutation ts(-) 296). Conditions leading to absence of repression in the mutant strain have been correlated with a sharp decrease in bulk tRNA(met) charging, whereas conditions which restore repressibility of group I enzymes also restore tRNA(met) charging. These findings implicate methionyl-tRNA in the regulatory process. However, the absence of a correlation in the wild type between methionyl-tRNA charging and the levels of methionine group I enzymes suggests that only a minor iso accepting species of tRNA(met) may be devoted with a regulatory function. Repressibility of the same four enzymes (group I) was also decreased in strains carrying the regulatory mutation eth2(r). Although structural genes coding for two of these enzymes, as well as mutations ts(-) 296 and eth2(r) segregate independently to each other, synthesis of group I enzymes is coordinated. The pleiotropic regulatory system involved seems then to comprise beside a "regulatory methionyl tRNA(met)," another element, product of gene eth2, which might correspond either to an aporepressor protein or to the "regulatory tRNA(met)" itself. Regulation of group II enzymes is defined by response to exogenous methionine, absence of response to either mutations ts(-) 296 and eth2(r), and absence of coordinacy with group I enzymes. However, the two enzymes which belong to this group and are both involved in threonine and methionine biosynthesis undergo distinct regulatory patterns. One, aspartokinase, is subject to a bivalent repression exerted by threonine and methionine, and the other, homoserine dehydrogenase, is subject only to methionine-mediated repression. Participation of at least another aporepressor and another corepressor, different from the ones involved in regulation of group I enzymes, is discussed.