Studies on nitrogen metabolism during somatic embryogenesis in carrot. I. Utilization of α-alanine as a nitrogen source

α-Alanine added to a culture medium was incorporated into cells and/or embryos during rapid cell proliferation and globular embryo formation, in which an active protein synthesis was occurring. After the incorporation into cells, α-alanine seemed to be quickly transformed to glutamic acid by alanine aminotransferase and utilized as a nitrogen source. Alanine aminotransferase activity was observed in cells and/or embryos, although the activity decreased during culture period. Utilization of α-alanine by cultured cells as a nitrogen source was discussed in relation to its stimulative effect on somatic embryogenesis.     

Biomass as a source of monomers: I — Synthesis of 2-vinylfuran

The condensation reaction between furfural and methyltriphenyl-phosphonium bromide was carried out using a solid-liquid transfer process in the presence of an excess of potassium carbonate. 2-vinylfuran was obtained selectively and quantitatively. Nitrobenzene was used as a solvent and 2-vinylfuran could thus be extracted readily; no polymerisation side reactions were involved.     

The C-terminal αI domain linker as a critical structural element in the conformational activation of αI integrins

The activation of α/β heterodimeric integrins is the result of highly coordinated rearrangements within both subunits. The molecular interactions between the two subunits, however, remain to be characterized. In this study, we use the integrin α(L)β(2) to investigate the functional role of the C-linker polypeptide that connects the C-terminal end of the inserted (I) domain with the β-propeller domain on the α subunit and is located at the interface with the βI domain of the β chain. We demonstrate that shortening of the C-linker by eight or more amino acids results in constitutively active α(L)β(2) in which the αI domain is no longer responsive to the regulation by the βI domain. Despite this intersubunit uncoupling, both I domains remain individually sensitive to intrasubunit conformational changes induced by allosteric modulators. Interestingly, the length and not the sequence of the C-linker appears to be critical for its functionality in α/β intersubunit communication. Using two monoclonal antibodies (R7.1 and CBR LFA-1/1) we further demonstrate that shortening of the C-linker results in the gradual loss of combinational epitopes that require both the αI and β-propeller domains for full reactivity. Taken together, our findings highlight the role of the C-linker as a spring-like element that allows relaxation of the αI domain in the resting state and controlled tension of the αI domain during activation, exerted by the β chain.     

Kinetics of α-amylase production in a batch and fed-batch culture of Bacillus subtilis with caseinate as nitrogen source and starch as carbon source

Summary Analysis of a large number of experimental data from the cultivation of Bacillus subtilis formed the basis for a kinetic model of the process explaining the effect of composition of the culture medium and of the growth rate on the rate of enzyme production. The resulting rate of formation of -amylase (EC 3.2.1.1) reflects the sum of the rate of enzyme production and the rate of its degradation as affected by the environment. The kinetic dependence confirms the previously described mechanism of regulation of enzyme biosynthesis. The mathematical model of the process served here to determine the optimal conditions for enzyme biosynthesis which were then verified in a fed-batch cultivation. The production of the enzyme in fed-batch culture was found to be twice that found in a batch cultivation.Symbols X biomass concentration, g·l^-1 - t time, h - S ₁ caseinate concentration, g·l^-1 - S ₂ starch concentration, g·l^-1 - P product concentration, U·ml^-1 - r _P specific rate of product formation, U·g^-1·h^-1 - R _P total rate of product formation, U·l^-1·h^-1 - Y yield coefficient - specific growth rate, h^-1     

Heterogeneity in tissue culture infection models: a source of novel host-pathogen interactions?

Tissue cultures have been successfully exploited to dissect cellular and molecular mechanisms of microbial infections. Most of the methods used in this model conclude with data describing host and pathogen 'average' responses. Microscopy, however, reveals that such interplay is very diverse and that both partners are composed of phenotypically heterogeneous populations. Thus, upon co-incubation in the plate assay, neither all cultured host cells are infected nor all pathogen cells inflict alterations in host physiology. Despite its obvious impact in data interpretation, the basis of this heterogeneity remains in most cases unknown. Addressing this issue is encouraging since may contribute to uncover novel interactions in the host-pathogen scenario.     

An untargeted metabolomic approach in the chemotaxonomic assessment of two Salvia species as a potential source of α-bisabolol

α-Bisabolol is a commercially important aroma chemical currently obtained from the Candeia tree (Vanillosmopsis erythropappa). Continuous unsustainable harvesting of the Candeia tree has prompted the urgent need to identify alternative crops as a source of this commercially important sesquiterpene alcohol. A chemotaxonomic assessment of two Salvia species indigenous to South Africa is presented and recommended as a potential source of α-bisabolol. The essential oil obtained by hydrodistillation of the aerial parts was analysed by gas chromatography coupled to mass spectrometry (GC–MS) and mid-infrared spectroscopy (MIRS). Orthogonal projections to latent structures–discriminant analysis (OPLS–DA) were used for multivariate classification of the oils based on GC–MS and MIRS data. Partial least squares (PLS) calibration models were developed on the MIRS data for the quantification of α-bisabolol using GC–MS as the reference method. A clear distinction between Salvia stenophylla and Salvia runcinata oils was observed using OPLS–DA on both GC–MS and MIRS data. The MIR calibration model showed high coefficient of determination (R² = 0.999) and low error of prediction (RMSEP = 0.540%) for α-bisabolol content.     

Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 2: α-Branched quaternary amines

We report the synthesis and biological evaluation of a series of novel α-branched pyrazinoyl quaternary amines for their ability to block ion transport via the epithelial sodium channel (ENaC) in human bronchial epithelial cells (HBECs). Compound 12 g has an IC(50) of 30 nM and is highly efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 1 μg kg(-1) at 1h. In addition the SAR results demonstrate for the first time the chiral nature of the binding site of human ENaC. As such, pyrazinoyl quaternary amines represent a promising new class of ENaC blockers for the treatment of cystic fibrosis that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride.     

Role of the side chain stereochemistry in the α-glucosidase inhibitory activity of kotalanol,a potent natural α-glucosidase inhibitor. Part 2

To examine the role of the side chain of kotalanol (2), a potent natural α-glucosidase inhibitor isolated from Salacia reticulata, on inhibitory activity, four diastereomers (11a–11d) with reversed configuration (S) at the C-4′ position in the side chain were synthesized and evaluated. Two of the four (11b and 11d) significantly lost their inhibitory activity against both maltase and sucrase, while the other two (11a and 11c) sustained the inhibitory activity to a considerable extent, showing distinct activity in response to the change of stereochemistry of the hydroxyls at the 5′and 6′ positions. Different activities were rationalized with reference to in silico docking studies on these inhibitors with hNtMGAM. Against isomaltase, all four analogs showed potent inhibitory activity as well as 2, and 11b and 11d exhibited enzyme selectivity.     

Disulfide bridge as a linker in nucleic acids’ bioconjugation. Part I: An overview of synthetic strategies

This 2-part article reviews methods of oligonucleotides functionalization with thiol tethers and their consecutive use in conjugation with other (macro)molecules via a disulfide bridge. This relatively inexpensive, robust and reversible method of conjugation of DNAs, RNAs and their analogs holds a prominent position in a modern biochemistry toolbox and therefore there is a wealth of literature on the subject. In part I methods of thiol/disulfide groups introduction into oligonucleotide strands have been systematized and discussed. A digest of conjugation methods is presented as well.     

Part II. Chiral dirhodium (II) catalysts with ortho-metalated arylphosphane ligands in the enantioselective intramolecular cyclopropanation of a racemic α-diazo ketone

In this report, chiral dirhodium (II) with ortho-metalated phosphane ligands, namely (M)-Rh₂(O₂CR) ₂(PC)₂ [PC = ortho-metalated aryl phosphane, O₂CR = carboxylate bridging ligands) (1a-g), have been used for the intramolecular cyclopropanation of racemic1-diazo-6-methyl-3-(2-propenyl)-5-hepten-2-one (2), containing both a tri- and monosubstituted carbon-carbon double bond, in pentane. The highest level of regiocontrol has been obtained with chiral catalyst Rh₂(O₂CCH₃)₂[(p-MeC₆H₃)P(p-MeC₆H₄)₂]₂ (M)-1c, affording favorably trisubstituted cyclopropane 3 versus monosubstituted cyclopropane 4 in 74:26 ratio. An exceptional diastereoselectivity was obtained with the entire catalyst series, leading to the unique formation of the syn products. Excellent enantiocontrol values (80-90% ee) have been achieved with catalysts 1a [(PC = (C₆H₃)P(C₆H₄)₂, R = C(CH₃)₃)], 1c [PC = p-MeC₆H₃)P(p-MeC₆H₄)₂, R = CH₃], 1f [PC = m-CH₃C₆H₃)P(m-CH₃C₆H₄)₂, R = CF₃] and 1g [PC = 3,5-(CH₃)₂C₆H₃)P(3,5-(CH₃)₂C₆H₄)₂, R = CF₃] at room temperature. Pentane is found to be a convenient solvent for high enantiocontrol in the cyclopropanation of α-diazo ketone 2.     

Reductive methylation as a probe of the heat-labile α-bungarotoxin-acetylcholine receptor membrane complex: Evidence for surface interactions

α-Bungarotoxin (α-Bgt) is a potent postsynaptic neurotoxin which blocks neurotransmission by binding very tightly to the acetylcholine-receptor (AcChR) protein. We have previously shown (P. Calvo-Fernandez, and M. Martinez-Carrion (1981) Arch. Biochem. Biophys., 208, 154–159) that α-Bgt free in its native solution conformation incorporates 12 methyl groups when reductively methylated using formaldehyde and sodium cyanoborohydride. We now show that when the α-Bgt molecule is bound to the AcChR contained in native membranes prepared from Torpedo californica electroplax, the number of accessible methylation sites is significantly reduced. This favors a model of α-Bgt-AcChR interaction involving significant numbers of lysyl moieties distributed over a reasonably large surface of the toxin molecule. In addition, this paper presents a novel procedure for the rapid and nondestructive dissociation of the toxin-AcChR membrane complex which takes advantage of the thermal instability of the complex.     

On the conformational dependence of the proton chemical shifts in nucleosides and nucleotides III. Proton chemical shifts of 5′-nucleotides as a function of different conformational parameters

The variation of the chemical shift of the protons of 5′-UMP and 5′-AMP is calculated as a function of χ_CN, ψ and ? torsion angles. The shift of H8 of 5′-AMP and H6 of 5′-UMP is found to be very sensitive to the value of χ_CN. For the anti conformations the shift of these protons is more sensitive to the value of the rotation about CS′-05′ than about C4′-CS′. For the protons of the ribose the calculations show that for the C2′-endo pucker H3′ and H2′ undergo the largest chemical shift variations when ? and ψ vary. The calculated variations are considered in relation with the role of the conformation of the nucleotides in the chemical shift variation between mono and polynucleotides and between the different helical structures of polynucleotides.     

Electrostatic and hydrophobic interactions of lipid-associated α-synuclein: The role of a water-limited interfaces in amyloid fibrillation

Human α‑synuclein (αSyn) is an intrinsically disordered protein (IDP) whose biological and pathological functions in brain neuronal cells have not yet been fully elucidated. αSyn intrinsically participates in aiding neurotransmitter trafficking through αSyn the association with lipid membranes. However, lipid-associated states of αSyn also induce amyloid self-assembly that is linked to the pathogenesis of various synucleinopathies. These contradicting actions arise from the limited water content near lipid-water interfaces that controls αSyn electrostatic and hydrophobic interactions. Thus, understanding the molecular interactions between αSyn and lipid membranes in the presence of water molecules is critical in elucidating the pivotal role of lipid-associated αSyn in amyloid self-assembly. In this review, we describe how the membrane interface controls electrostatic and hydrophobic interactions of lipid-associated αSyn. Moreover, membrane amyloid self-assembly of αSyn will be further discussed with regards to the structural dynamics of lipid-associated αSyn and water molecules near the interface.     

Discovery of imidazo[1,2-b]pyridazines as IKKβ inhibitors. Part 2: improvement of potency in vitro and in vivo

We have increased the potency of imidazo[1,2-b]pyridazine derivatives as IKKβ inhibitors with two strategies. One is to enhance the activity in cell-based assay by adjusting the polarity of molecules to improve permeability. Another is to increase the affinity for IKKβ by the introduction of additional substituents based on the hypothesis derived from an interaction model study. These improved compounds showed inhibitory activity of TNFα production in mice.     

Identifying functionally important conformational changes in proteins: activation of the yeast α-factor receptor Ste2p

We have developed a procedure in which disulfide cross-links are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast α-mating pheromone receptor Ste2p, a member of the superfamily of G protein-coupled receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cysteines. Libraries of mutated receptors were then screened for alleles that exhibit constitutive signaling. Two strongly activated alleles were recovered containing cysteine residues in transmembrane (TM) segments 5 and 6. Constitutive activity of these alleles was dependent on the presence of both introduced cysteines and was sensitive to reducing agent. Cross-linked peptides derived from the mutant receptors were detected by immunoblotting. Additional sites of cross-linking between TM segments 5 and 6 that did not lead to constitutive activation were also identified. These results indicate that relative motion of the TM segments 5 and 6 in the extracellular half of the membrane is sufficient to activate the receptor and that TM segment 6, but not TM segment 5, exhibits rotational mobility that is not associated with receptor activation.     

2-Pyridylmetallocenes: Part I. Electrophilic halogenation of 2-pyridylferrocene. Molecular structures of 2-pyridylferrocene and its α-brominated and -fluorinated derivatives. Synthesis of 2-pyridylruthenocene and 2-pyridylcymantrene

A new high-yield synthesis of 2-pyridylferrocene (1) without formation of the 1,1′-disubstituted product has been developed. Also the corresponding ruthenocene and cymantrene derivatives [C₅H₄(2-C₅H₄N)]ML_n (ML_n = Ru(C₅H₅) (2), Mn(CO)₃ (3)) were prepared and fully characterized. Ortho-lithiation of 1 followed by electrophilic halogenation yielded [C₅H₃X(2-C₅H₄N)]Fe(C₅H₅) [X = F (4), Cl (5), Br (6), I (7)], with 4 only being the second reported and first fully characterized fluoroferrocene. The molecular structures of 1, 4 and 6 have been determined by X-ray crystallography.     

Proteases as catalysts of enantioselective α-amino ester hydrolysis: 2. Pronase-catalysed hydrolysis of tryptophan methyl ester

The influence of pH, temperature, EDTA and metal ions on the rate of hydrolysis of methyl esters of d- and l-tryptophan by pronase from Streptomyces griseus has been studied. pH, Ca²⁺ and Co²⁺ ions have different effects on the hydrolysis of d- and l-substrates, which permits the enantioselectivity of the reaction to be regulated. Maximum enantioselectivity was observed at pH 7 in the presence of 10^?3–10^?2m Co²⁺. The data obtained are compared with the literature data for the hydrolysis of other low molecular weight substrates by pronase. It is suggested that leucine aminopeptidase makes a considerable contribution to the observed esterase activity of pronase.     

Kinetics and mechanism of G-quadruplex formation and conformational switch in a G-quadruplex of PS2.M induced by Pb²⁺

DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG(3)TAG(3)CG(3)TTG(2)). UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb(2+) ions to PS2.M is two, suggesting that the Pb(2+) ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO(3))(2) to the Na(+)- or K(+)-induced G-quadruplexes, which may be attributed to the replacement of Na(+) or K(+) by Pb(2+) ions and the generation of a more compact Pb(2+)-PS2.M structure. Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.