Knoevenagel condensation of 2-carbethoxycyclohexanone and malononitrile: Synthesis of 4-cyano-3-ethoxy-1-hydroxy-5,6,7,8-tetrahydroisoquinoline, an isomer of the abnormal product

The structure of the abnormal product 1a formed in the Knoevenagel condensation of 2-carbethoxycyclohexanone and malononitrile has been further confirmed. Oxidation of the tetrahydroisoquinoline 3b using Na2Cr2O-AcOH-H2SO4 gave the keto isoquinoline 3d and the isoquinoline-1-carboxylic acid 5a. The acid chloride of 5a was condensed with diethyl ethoxymagnesiomalonate to afford after decarbethoxylation the methyl ketone 5d which on Baeyer-Villiger oxidation gave a mixture of the acetate 1g and the title compound 1b. The unambiguous synthesis of 1b confirms the structure assigned earlier to the title compound also formed during the partial hydrolysis of the diethoxy compound 1c. Condensation of 2-acetylcyclohexane-1,3-dione with malononitrile gave the quinoline derivative 4c which on ethylation yielded the ketoquinoline 4d. The present studies have confirmed that the quinoline compound 4a is also formed in the condensation of 2-acetylcyclohexanone and cyanoacetamide.

Positive associations of serum Perfluoroalkyl substances with uric acid and hyperuricemia in children from Taiwan

To investigate the risk of hyperuricemia in relation to Perfluoroalkyl substances (PFASs) in children from Taiwan, 225 Taiwanese children aged 12-15 years were recruited from 2009 to 2010. Linear and logistic regression models were employed to examine the influence of PFASs on serum uric acid levels. Findings revealed that eight of ten PFASs analyses were detected in > 94% of the participants' serum samples. Multivariate linear regression models revealed that perfluorooctanic acid (PFOA) was positively associated with serum uric acid levels (β=0.1463, p<0.05). Of all the PFASs analyses, only PFOA showed a significant effect on elevated levels of hyperuricemia (aOR=2.16, 95%CI: 1.29-3.61). When stratified by gender, the association between serum PFOA and uric acid levels was only evident among boys (aOR=2.76, 95%CI: 1.37-5.56). In conclusion, PFOA was found to be associated with elevated serum levels of uric acid in Taiwanese children, especially boys. Further research is needed to elucidate these links.

Effect of depletion of vitamin A, followed by supplementation with retinyl acetate or retinoic acid, on regeneration of rat liver

After partial hepatectomy the net increase in tissue weight and in RNA, DNA and proteins in the regenerating liver was markedly less in vitamin A-deleted or retinoic acid-supplemented male rats, compared with the corresponding normal control or retinyl acetate-supplemented ones.

Mechanism of aromatic hydroxylation *1, , *2, , *3: Properties of a model for pyridine nucleotide-dependent flavoprotein hydroxylases

A model (NADH-phenazine methosulfate-O2) formally similar to pyridine nucleotide-dependent flavoprotein hydroxylases catalyzed the hydroxylation of several aromatic compounds. The hydroxylation was maximal at acid pH and was inhibited by ovine Superoxide dismutase, suggesting that perhydroxyl radicals might be intermediates in this process. The stoichiometry of the reaction indicated that a univalent reduction of oxygen was occurring. The correlation between the concentration of semiquinone and hydroxylation, and the inhibition of hydroxylation by ethanol which inhibited semiquinone oxidation, suggested the involvement of phenazine methosulfate-semiquinone. Activation of hydroxylation by Fe3+ and Cu2+ supported the contention that univalently reduced species of oxygen was involved in hydroxylation. Catalase was without effect on the hydroxylation by the model, ruling out H2O2 as an intermediate. A reaction sequence, involving a two-electron reduction of phenazine methosulfate to reduced phenazine methosulfate followed by disproportionation with phenazine methosulfate to generate the semiquinone, was proposed. The semiquinone could donate an electron to O2 to generate O2 which could be subsequently protonated to form the perhydroxyl radical.

Starch composites with aconitic acid

The aim of this project is to examine the effectiveness of using aconitic acid (AcA), a tricarboxylic acid which contains a carbon/carbon double bond (C=C), to enhance the properties of starch-based films. Starch/glycerol cast films were prepared with 0, 2, 5, 10 and 15 wt% AcA (starch wt% basis) and the properties analysed. It was shown that AcA acted as both a cross-linking agent and also a strong plasticising agent. The 5 wt% AcA derived starch films were the most effectively cross-linked having the lowest solubility (28 wt%) and decreased swelling coefficient (35 vol.%) by approximately 3 times and 2.4 times respectively compared to the control film submerged in water (23 °C). There was also a significant increase in the film elongation at break by approximately 35 times (compared to the control) with the addition of 15 wt% AcA, emphasising the plasticising effect of AcA. However, generally there was a reduced tensile strength, softening of the film, and reduced thermal stability with increased amounts of AcA.

Rate of ribonucleic acid chain growth in Mycobacterium tuberculosis H37Rv.

Two methods were employed to measure the rate of ribonucleic acid (RNA) chain growth in vivo in Mycobacterium tuberculosis H37Rv cultures growing in Sauton medium at 37 degrees C, with a generation time of 10 h. In the first, the bacteria were allowed to assimilate [3H]uracil or [3H]guanine into their RNA for short time periods. The RNA was then extracted and hydrolyzed with alkali, and the radioactivity in the resulting nucleotides and nucleosides was measured. The data obtained by this method allowed the calculation of the individual nucleotide step times during the growth of RNA chains, from which the average rate of RNA chain elongation was estimated to be about 4 nucleotides per s. The second method employed the antibiotic rifampin, which specifically inhibits the initiation of RNA synthesis without interfering with the elongation and completion of nascent RNA chains. Usint this method, the transcription time of the 16S, 23S, and 5S ribosomal RNA genes was estimated to be 7.6 min, which corresponds to a ribosomal RNA chain growth rate of 10 nucleotides per s.

Conformation of polypeptide-chains containing both l-residues and D-residues .2. Double-helical structures of poly-ld-peptides

Polypeptides with alternating L- and D-amino acid residues can take up stereochemically satisfactory coaxial double-helical structures, both antiparallel and parallel, which are stabilized by systematic interchain NH O hydrogen bonds. Semiempirical energy calculations over allowed regions of conformational space have yielded the characteristics of these double-helices. There are four possible types of antiparallel double-helices - A3, A4, A5 and A6, with n, the number of LD peptide units per turn, around 2.8, 3.6, 4.5 and 5.5 respectively, while for the parallel double-helices there are two types, P3 and P4, having similar helical parameters as in A3 and A4. The hydrogen-bonding scheme restricts the pitch in all the models to the narrow range of 10.0 to 11.5 Å. All these helices have large central cores whose radii increase proportionately with n. In this respect, A3 and A4 are suitable models for the structure of gramicidin A. In terms of their relative energies, antiparallel double-helices are marginally more stable than those with parallel strands. Our results indicate that the energy differences amongst the members in the antiparallel family are not significant and thus provide an explanation for the polymorphism reported for poly(γ-benzyl-LD-glutamate).

At4g24160, a Soluble Acyl-Coenzyme A-Dependent Lysophosphatidic Acid Acyltransferase

Human CGI-58 (for comparative gene identification-58) and YLR099c, encoding Ict1p in Saccharomyces cerevisiae, have recently been identified as acyl-CoA-dependent lysophosphatidic acid acyltransferases. Sequence database searches for CGI-58 like proteins in Arabidopsis (Arabidopsis thaliana) revealed 24 proteins with At4g24160, a member of the alpha/beta-hydrolase family of proteins being the closest homolog. At4g24160 contains three motifs that are conserved across the plant species: a GXSXG lipase motif, a HX4D acyltransferase motif, and V(X)(3)HGF, a probable lipid binding motif. Dendrogram analysis of yeast ICT1, CGI-58, and At4g24160 placed these three polypeptides in the same group. Here, we describe and characterize At4g24160 as, to our knowledge, the first soluble lysophosphatidic acid acyltransferase in plants. A lipidomics approach revealed that At4g24160 has additional triacylglycerol lipase and phosphatidylcholine hydrolyzing enzymatic activities. These data establish At4g24160, a protein with a previously unknown function, as an enzyme that might play a pivotal role in maintaining the lipid homeostasis in plants by regulating both phospholipid and neutral lipid levels.

Conformational Choices for the Stereochemically Constrained gamma-Amino Acid Residue Gabapentin: Theoretical Studies and Correlation With Experimental Results

Gabapentin (1-aminomethylcyclohexaneacetic acid, Gpn) is an achiral, conformationally constrained gamma amino acid residue. A survey of available crystal structures of Gpn peptides reveals that the torsion angles about the C-gamma-C-beta (theta(1)) and C-beta-C-alpha(theta(2)) bonds are overwhelmingly limited to gauche, gauche (g(+)g(+)/g(-)g(-)) conformations. The Gpn residue forms C-7 and C-9 hydrogen bonds in which the donor and acceptor atoms come from the flanking peptide units. In combination with alpha amino acid residues alpha gamma and gamma alpha segments can adopt C-12 hydrogen bonded structures. The conformational choices available to the Gpn residue have been probed using energy calculations, adopting a grid search strategy. Ramachandran phi-psi maps have been constructed for fixed values of theta(1) and theta(2), corresponding to the gauche and trans conformations. The sterically allowed and energetically favorable regions of conformational space have been defined and experimental observations compared. C-7 and C-9 hydrogen bonded conformational families have been identified using a grid search approach in which theta(1) and theta(2) values are varied over a range of +/- 10 degrees about ideal values at 1 degrees intervals. The theoretical analysis together with experimental observations for 59 Gpn residues from 35 crystal structures permits definition of the limited range of conformational possibilities at this gamma amino acid residue. .

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.

Stereochemical restrictions on the occurrence of amino acid residues in the collagen structure

The primary structure of collagen is characterized by the repeating tripeptide sequence (Gly-R2-R3)n. The results of theoretical studies, carried out using contact criteria to compute the stereochemically allowed orientations for various side chains at locations 2 and 3, are reported here. It is found that side chains with only γ-atoms, as in valine, serine and threonine, or with only one δ-methyl group, as in isoleucine, can occur equally well at locations 2 and 3, as is actually the case in collagen. Side chains with two Cδ-atoms, as in leucine and phenyl-alanine, can also be accommodated at both positions. However, if they occur as R3 their freedom of orientation is severely restricted in the presence of a proline residue as R2 in a neighbouring chain. If water molecules bound to the chains of the triple helix are assumed to be present, then location 3 is virtually impossible for leucine and phenylalanine residues. Location 2 is, however, unaffected, and their presence as R2 can help to shield the water molecules from disturbance by the solvent medium. This may be the reason for the preferential occurrence of Leu and Phe residues in location 2 in the collagen triplets, although the polypeptides (Gly-Pro-Leu)n and (Gly-Pro-Phe)n form collagen-like structures.

Mechanism of action of selenious acid in the electrodeposition of manganese

The effect of selenious acid as an addition agent in the electrodeposition of manganese was studied by analysing the current-potential curves for manganese deposition. The mechanism of action of this addition agent was found to be essentially similar to that proposed for sulphur dioxide, namely to affect the manganese deposition indirectly by influencing the hydrogen evolution reaction which is a parallel reaction at the electrode surface.

X-ray studies on crystalline complexes involving amino acids and peptides. XXIII. Variability in ionization state, conformation and molecular aggregation in the complexes of succinic acid with DL- and L-lysine

Crystalline complexes of succinic acid with DL- and L-lysine have been prepared and analysed by X-ray diffraction. DL-Lysine complex: C6HIsN202 + 1 2- 1 ~C4H404 .~C4H604, Mr -- 264"2, PI, a = 5"506 (4), =8.070(2), c=14.089(2) A,, a=92.02(1), /3= 100"69 (3), y = 95"85 (3) ~>, Z = 2, Dx = 1"44 g cm -3, R = 0.059 for 2546 observed reflections. Form I of the e-lysine complex: C6HIsN20-, ~ .C4H504, Mr = 264.2, P1, a = 5" 125 (2), b = 8"087 (1), c = 8"689 (1) A,, a = 112.06 (1), /3 = 99.08 (2), y = 93"77(2) °, Z--l, D,,,=1"34(3), Dx=l"34gcm 3 R = 0.033 for 1475 observed reflections. Form II of + I 2- the e-lysine complex: C6H15N202 .,iC4H404 .- 1 I ") 4C4H604.4(C4HsO4""H'"CaH404)" , Mr = 264"2, P1, a = 10.143 (4), b = 10.256 (2), c = 12"916 (3) A,, a = 105.00 (2),/3 = 99-09 (3), y = 92"78 (3)::, Z = 4, Dm= 1"37(4), D,.= 1.38gcm 3, R=0.067 for 2809 observed reflections. The succinic acid molecules in the structures exhibit a variety of ionization states. Two of the lysine conformations found in the complexes have been observed for the first time in crystals containing lysine. Form II of the L-lysine complex is highly pseudosymmetric. In all the complexes, unlike molecules aggregate into separate alternating layers. The basic element of aggregation in the lysine layer in the complexes is an S2-type head-to-tail sequence. This element combines in different ways in the three structures. The basic element of aggre gation in the succinic acid layer in the complexes is a hydrogen-bonded ribbon. The ribbons are interconnected indirectly through amino groups in the lysine layer.

A comparative study of the early effects of phenobarbital and 3-methylcholanthrene on the synthesis and transport of ribonucleic acid in rat liver.

At 2-3 h after phenobaribtal administration, the drug has no effect on nucleoplasmic RNA synthesis and decreases nucleolar RNA synthesis. However, at this time there is an increase in the labelling of cytoplasmic poly(A)-containing RNA, even though there is decreased labelling of total polyribosomal RNA. The decrease in labelling of nucleolar and total polyribosomal RNA owing to phenobarbital is a transient phenomenon. Under similar conditions, 3-methylcholanthrene has no effect on nucleolar RNA synthesis, but leads to an increase in synthesis of nucleoplasmic and cytoplasmic poly(A)-containing RNA. Cytosol isolated from phenobarbital-treated, but not from 3-methyl-cholanthrene-treated, animals facilitates an enhanced transport of RNA from nuclei. At the time points investigated, 3-methylcholanthrene or its metabolite shows a 10-15-fold higher concentration in the chromatin than that of phenobarbital or its metabolite. It is suggested that the primary effect of phenobarbital is at the cytoplasmic level, promoting the transport of RNA from the nuclei, which can act as a trigger for enhanced transcription at later periods. 3-Methylcholanthrene or its metabolite directly binds to the chromatin and evokes a selective transcriptional response.

Metal-Nucleotide Interactions: Crystal Structures of Alkali (Li+, Na+, K+) and Alkaline Earth (Ca2+, Mg2+) Metal Complexes of Adenosine 2'-Monophosphate

Crystal structures of lithium, sodium, potassium, calcium and magnesium salts of adenosine 2'-monophosphate (2'-AMP) have been obtained at atomic resolution by X-ray crystallographic methods. 2'-AMP.Li belongs to the monoclinic space group P21 with a = 7.472(3)Å, b = 26.853(6) Å, c = 9.184(1)Å, b = 113.36(1)Å and Z= 4. 2'-AMP.Na and 2'-AMP.K crystallize in the trigonal space groups P31 and P3121 with a = 8.762(1)Å, c = 34.630(5)Å, Z= 6 and a = 8.931(4), Åc = 34.852(9)Å and Z= 6 respectively while 2'-AMP.Ca and 2'-AMP.Mg belong to space groups P6522 and P21 with cell parameters a = 9.487(2), c = 74.622(13), Z = 12 and a = 4.973(1), b = 10.023(2), c = 16.506(2), beta = 91.1(0) and Z = 2 respectively. All the structures were solved by direct methods and refined by full matrix least-squares to final R factors of 0.033, 0.028, 0.075, 0.069 and 0.030 for 2'-AMP.Li, 2'-AMP.Na, 2'- AMP.K, 2'-AMP.Ca and 2'-AMP.Mg, respectively. The neutral adenine bases in all the structures are in syn conformation stabilized by the O5'-N3 intramolecular hydrogen bond as in free acid and ammonium complex reported earlier. In striking contrast, the adenine base is in the anti geometry (cCN = -156.4(2)°) in 2'-AMP.Mg. Ribose moieties adopt C2'-endo puckering in 2'-AMP.Li and 2'-AMP.Ca, C2'-endo-C3'-exo twist puckering in 2'-AMP.Na and 2'-AMP.K and a C3'-endo-C2'-exo twist puckering in 2'-AMP.Mg structure. The conformation about the exocyclic C4'-C5' bond is the commonly observed gauche-gauche (g+) in all the structures except the gauche- trans (g-) conformation observed in 2'-AMP.Mg structure. Lithium ions coordinate with water, ribose and phosphate oxygens at distances 1.88 to 1.99Å. Na+ ions and K+ ions interact with phosphate and ribose oxygens directly and with N7 indirectly through a water oxygen. A distinct feature of 2'-AMP.Na and 2'-AMP.K structures is the involvement of ribose O4' in metal coordination. The calcium ion situated on a two-fold axis coordinates directly with three oxygens OW1, OW2 and O2 and their symmetry mates at distances 2.18 to 2.42Å forming an octahedron. A classic example of an exception to the existence of the O5'-N3 intramolecular hydorgen bond is the 2'-AMP.Mg strucure. Magnesium ion forms an octahedral coordination with three water and three phosphate oxygens at distances ranging from 2.02 to 2.11Å. A noteworthy feature of its coordination is the indirect link with N3 through OW3 oxygen resulting in macrochelation between the base and the phosphate group. Greater affnity of metal clays towards 5' compared to 2' and 3' nucleotides (J. Lawless, E. Edelson, and L. Manring, Am. Chem. Soc. Northwest Region Meeting, Seattle. 1978) due to macrochelation infered from solution studies (S. S. Massoud, H. Sigel, Eur. J. Biochem. 179, 451-458 (1989)) and interligand hydrogen bonding induced by metals postulated from metal-nucleotide structures in solid state (V. Swaminathan and M. Sundaralingam, CRC. Crit. Rev. Biochem. 6, 245-336 (1979)) are borne out by our structures also. The stacking patterns of adenine bases of both 2'-AMP.Na and 2'-AMP.K structures resemble the 2'-AMP.NH4 structure reported in the previous article. 2'-AMP.Li, 2'-AMP.Ca and 2'-AMP.Mg structures display base-ribose O4' stacking. An overview of interaction of monovalent and divalent cations with 2' and 5'-nucleotides has been presented.