Novel taxa-4(20),12-diene and 2(3→20)abeotaxane from needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Novel Taxa-4(20),12-diene and 2(3→20)Abeotaxane from Needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Biotransformation of the diterpene 15β-hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene by the fungus Fusarium fujikuroi

15β-Hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene (4) was biotransformed by the fungus Fusarium fujikuroi into 3α,11β,15β-trihydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene (5). The hydroxylation at C-3(α) in this diterpene reminds a similar reaction that occurs at C-13 in the biosynthesis of gibberellic acid in this fungus. The presence of the 15β-alcohol in the substrate directs the second hydroxylation at C-11(β), which had been observed in the incubation of ent-kaur-16-ene derivatives with this fungus when the C-19 hydroxylation was inhibited by the existence in the molecule of a 3α-OH or 3-oxo group. We also show that the angelate of the substrate is an undescribed natural product now identified as a component of the plant Distichoselinum tenuifolium.     

Cloning and mapping of bovine ZFX gene to the long arm of the X-chromosome (Xq34) and homologous mapping of ZFY gene to the distal region of the short arm of the bovine (Yp13), ovine (Yp12–p13), and caprine (Yp12–p13) Y Chromosome

A part of mouse Zfy-2 sequence was synthesized and used to screen a genomic library of the spinous country-rat (Tokudaia osimensis spp., 2n = 45). An isolated clone had the C-terminal region of Zfy, which consisted of 1190 bp, encoded 336 amino acid residues, and harbored 11 out of 13 zinc finger motifs. With this as a probe, a bovine testis cDNA library was screened. Two ZFX clones were isolated and their sequences combined. The short sequence, lacking part of the 5′ upstream region, was amplified by PCR or RT-PCR, cloned, and sequenced. A full-length ZFX was constructed by combining these three sequences. The bovine ZFX consisted of 5328 bp and encoded 800 amino acid residues, which contained 13 zinc finger motifs. ZFX was used as a probe for fluorescence in situ hybridization and was mapped to Xq34, different from its previously reported site at Xq21-q231. A SINE (short interspersed nuclear element) sequence consisting of 188 bp was found close to the end of the 3′-untranslated region of ZFX. The SINE sequence hybridized to all bovine chromosomes. ZFY is highly homologous with ZFX and, as a result, ZFY could be mapped simultaneously. ZFY was mapped to the distal region of the short arm of the Y Chromosome (Chr) (Yp13), contradicting the previously reported position Yq1. Ovine and caprine ZFY were also mapped with bovine ZFX. Both were mapped to the distal region of the short arm of the Y Chr (Yp12-p13). Ovine ZFX was mapped to a region close to the centromere of the X Chr (Xq13). Received: 23 July 1997 / Accepted: 30 September 1997     

IR and Raman study on the interactions of the 5′-GMP and 5′-CMP phosphate groups with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III)

The chief motive behind this research is the interest provoked by the presence of metal ions as necessary stabilizers of the negative charges of phosphate groups in nucleic acids. The effect that the presence of different metal ions produces on the band principally assigned to the nu(s) PO(3)(2-) mode has been studied using FT-IR and FT-Raman spectroscopy. The results obtained reveal the diagnostic capacity of these techniques in determining the type of metal ion interaction with respect to the mononucleotides that form DNA and RNA, providing a tool for improving the knowledge of the stabilizing or destabilizing effects of these ions on such macromolecules. The metal complexes of the ribonucleotides 5'-CMP and 5'-GMP with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III) were obtained in this study. After studying and analyzing the IR and Raman spectra of all these complexes and comparing them with the spectra of the corresponding disodium salts, it was verified that, independently of the type of nucleotide involved, the presence of the metal in the vicinity of the phosphate group produces an alteration in the aforementioned nu(s) PO(3)(2-) band. This effect is related to the type of interaction that the phosphate group has with the metal. Three components are observed: (1) one near 983-975 cm(-1) (detectable in IR and Raman), associated with phosphate groups in an electrostatic type of interaction with the metal ion, separated by two or more water molecules; (2) another near 989-985 cm(-1) (only in IR), associated with phosphate groups in indirect interaction through the water molecules of the coordination sphere of the metal ions; and (3) the IR and Raman bands near 1014-1001 cm(-1), which represent phosphate groups directly bonded to the metal ion. These results are supported by the behavior of 5'-CMP in aqueous solution in the presence of Mg(II) ions.     

A newborn child with karyotype 47,XX,+der(12) (12pter→12q12::8q24→8qter),t(8;12) (q24;q12) pat

Summary A case of Meckel or Gruber syndrome is reported, together with a survey of the relevant literature of recent years (1971–1977), in reference to a probably autosomal recessive inheritance of this malformation.     

Biotransformation of (-)-(R)-α-Phellandrene: Antimicrobial Activity of Its Major Metabolite

Terpene derivatives converted by microbial biotransformation constitute an important resource for natural pharmaceutical, fragrance, and aroma substances. In the present study, the monoterpene α-phellandrene was biotransformed by 16 different strains of microorganisms (bacteria, fungi, and yeasts). The transformation metabolites were initially screened by TLC and GC/MS, and then further characterized by NMR spectroscopic techniques. Among the six metabolites characterized, 6-hydroxypiperitone, α-phellandrene epoxide, cis-p-menth-2-en-1-ol, and carvotanacetone, which originated from (-)-(R)-α-phellandrene, are reported for the first time in this study. Additionally, the substrate and the metabolite 5-p-menthene-1,2-diol were subjected to in vitro antibacterial and anticandidal tests. The metabolite showed moderate-to-good inhibitory activities (MICs=0.125 to >4?mg/ml) against various bacteria and especially against Candida species in comparison with its substrate (-)-(R)-α-phellandrene and standard antimicrobial agents.     

4α-Methyl-5α-cholest-8(14)-en-3β-ol from the seeds of Capsicum annuum

A 4α-methylsterol was isolated from the seeds of Capsicum annuum and was identified as 4α-methyl-5α-cholest-8(14)-en-3β-ol. This seems to b     

Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12

Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.     

Biotransformation of 17α-ethynyl substituted steroidal drugs with microbial and plant cell cultures: A review

Structural modification of steroids through whole-cell biocatalysis is an invaluable procedure for the production of active pharmaceutical ingredients (APIs) and key intermediates. Modifications could be carried out with regio- and stereospecificity at positions hardly available for chemical agents. Much attention has been focused recently on the biotransformation of 17α-ethynyl substituted steroidal drugs using fungi, bacteria and plant cell cultures in order to obtained novel biologically active compounds with diverse structure features. Present article includes studies on biotransformation on 17α-ethynyl substituted steroidal drugs using microorganisms and plant cell cultures. Various experimental and structural elucidation methods used in biotransformational processes are also highlighted.     

Complexes of Co(II), Ni(II), Cu(II) and Zn(II) with 3′AMP and 2′AMP

Derivatives of Co(II), Ni(II), Cu(II) and Zn(II) with 3′AMP and 2′AMP were synthesized and characterized by IR UV-Vis and fluorescence spectroscopy. There seems to be bonding of the metal ion to the base in all cases. The activation test, using the complexes as allosteric labels, was carried out with rabbit muscle glycogen phosphorylase b, but the enzyme was not activated, confirming that the phosphate group must necessarily be bonded to position 5′ of the ribose in order to activate this enzyme.     

Expression of receptors for melatonin (MT1), thyroid hormone (TR-α), deiodinase (Dio-2), glucose transporters (GLUT-1 &4) and its relation with splenic cell survival (Bcl-2) of golden hamster,Mesocricetus auratus

Seasonal breeder utilises photoperiod as environmental clue to adjust their energetically demanding phenomenon such as reproduction and immunity by the mechanism of trade-off. The photoperiodic modulation of melatonin (MT-1)-thyroid hormone receptor (TR-α), deiodinase (Dio-2) activity and its interrelationship with glucose transporters (GLUT-1&4) in lymphoid organ of seasonal breeder is lacking that may explain possible role of photoperiod and its relationship with the cell survival factors (Bcl-2) in spleen of golden hamster, Mesocricetus auratus. We reported that photoperiod regulates the circulatory melatonin and thyroid hormone levels. Short-day-induced melatonin can act via MT1 and may enhance the expressions of GLUT-1&4 thereby the energy and cell survival factor (Bcl-2) in spleen. On the other hand, long-day-induced thyroid hormone is converted to bioactive from (T-4 to T3) by action of Dio-2 that acts through TR-α to maintain minimum level of energy for immune responses. In conclusion, present result explains the reason behind the basic molecular events involved in trade-offs mechanism in seasonal variation of immune responses.     

Levels of Gsα(short and long), Gα(olf) and Gβ(common) subunits, and calcium-sensitive adenylyl cyclase isoforms (1, 5/6, 8) in post-mortem human brain caudate and cortical membranes: comparison with rat brain membranes and potential stoichiometric relationships

The levels of expression of Gsα(short and long), Gα(olf) and Gβ(common) subunits, and calcium-sensitive adenylyl cyclases isoforms (AC1, 5/6, and 8) in human brain cortical and caudate membranes were quantified by western blot analysis in order to establish their contribution to the patterns of AC functioning. Both areas expressed Gsα(long) (52 kDa) with values ranging from about 1400 ng/mg of membrane protein in cerebral cortex to close to 600 ng/mg of membrane protein in caudate nucleus. In contrast, Gsα(short) and Gsα(olf) were expressed separately, Gsα(short) in cortical membranes with values around 500 ng/mg of membrane protein and Gα(olf) in caudate membranes with values around 1300 ng/mg of membrane protein. Quantitative measurements of Gβ, revealed a similar expression level in cortical and caudate membranes (5444±732 versus 5511±394 ng/mg protein; p=0.966). The B(max) values of GTPγS-dependent [(3)H]-forskolin binding show the following descending order: rat striatal membranes>rat cortical membranes=human caudate membranes>human cortical membranes. Therefore, as measured immunochemically and by [(3)H]-forskolin binding, there seems to be a vast excess of Gsα subunits over catalytic units of AC. The highest levels of AC5/6 expression were detected in caudate membranes. AC8 was little expressed, and there were no significant differences in the relative values between both human brain regions. Finally, the levels of the AC1 isoform were significantly lower in caudate than in cortical membranes. It is concluded that these stoichiometric data contribute nonetheless to explain the significant differences observed in signalling capacities through the AC system in both human brain regions.     

Oxidation of 17α,20β- and 17α,20α-Dihydroxypregn-4-en-3-ones,Side Products of Progesterone Biotransformation with Recombinant Microorganisms Expressing Cytochrome P-45017α

Progesterone biotransformation with recombinant yeasts Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 expressing bovine adrenocortical cytochrome P-45017 yielded 17-hydroxyprogesterone and two diols, 17,20- and 17,20-dihydroxypregn-4-en-3-ones. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17–20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17-hydroxylation and 20- and 20-reduction. The results widen the possibilities of enzymatic and chemical modifications of steroids.     

Synthesis of new chiral (η6-arene)(η4-diene)ruthenium(O) complexes

New Ru(η⁶-arene)(η⁴-diene) complexes, containing chiral substituents on the aromatic ring, have been prepared and characterized. The ¹H NMR inequivalence of the ortho and meta protons of the phenyl ring is discussed.     

Preparation and characterization of oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) complexes of 3,3′-(1,2-phenylenediimino)diacrolein

The oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) of 3,3′-(1,2-phenylenediimino)diacrolein were prepared and investigated by means of mass, electronic, vibrational, NMR and ESR spectroscopy as well as magnetic susceptibility measurements. The acetatomanganese(III) and chloroiron(III) complexes were confirmed to be of high spin type. The absorption bands appearing in the energy range greater than 23 000 cm⁻¹ were attributed to π→π^* transitions within a ligand molecule and charge- transfer transitions from metal to ligand. The metal complexes assume the square-planar configuration type since the ligand-field bands were detected in the 12 700–18 500 cm⁻¹ region. Strong bands appearing at 1601 and 1627 cm⁻¹ were assigned to the CC and CO stretching vibrational modes, respectively, and were shifted to lower frequency upon metal-coordination. A VO stretching band was observed at 982 cm⁻¹ for the oxovanadium(IV) complex and a CO stretching band was observed at 1547 cm⁻¹ for the acetatomanganese(III) complex. Upon complex formation the amine proton signal is found to vanish and the aldehydic methine proton signal in the lowest field is shifted upfield for the nickel(II), zinc(II) and palladium(II) complexes. ¹³C NMR spectra support the coordination structure of the complexes which is revealed by ¹H NMR spectra. As judged by the spin Hamiltonian parameters, the oxovanadium(IV) complex is of a square- planar type with an unpaired electron in the d_xy orbital and the copper(II) complex assumes a distorted square-planar coordination due to the presence of five- and six-membered chelate rings with an unpaired electron in the d_x2−y2 orbital.     

Molecular mechanics calculations of dA12·dT12 and of the curved molecule d(GCTCGAAAAA)4·d(TTTTTCGAGC)4

Using the AMBER software package (Weiner and Kollman 1981) substantially modified for electrostatic contributions, the structural energies of the double-stranded oligonucleotides dA₁₂·dT₁₂ and d(GCTCGAAAAA)₄·d(TTTTTCGAGC)₄ were minimized. Using various starting structures for the molecule dA₁₂·dT₁₂, one final structure is obtained which possesses the experimentally determined properties of poly(dA)·poly(dT). This structure is an A-form-B-form-hybrid structure similar to that of Arnott et al. (1983). The dA-strand is similar to an A-form while the dT-strand is similar to normal B-form. This structure and separately optimized B-form sequence stretches were used to construct the double-stranded fragment d(GCTCGAAAAA)₄ which again was optimized. This sequence, when imbedded in a DNA fragment as contiguous repeats, shows a gel migration anomaly which has been interpreted as stable curvature of the DNA (Diekmann 1986). The calculated structure of this sequence indeed has a curved helix axis and is discussed as a model for curved DNA. A theoretical formalism is presented which allows one to calculate the structural parameters of any nucleic acid double helix in two different geometrical representations. This formalism is used to determine the parameters of the base-pair orientations of the curved structure in terms of wedge as well as cylindrical parameters. In the structural model presented here, the curvature of the helix axis results from an alternation of two different DNA structures in which the base-pairs possess different angles with the helix axis (cylinder tilt). Resulting from geometric restraints, a negative cylinder tilt angle correlates strongly with the closing of the minor groove (wedge roll). The blocks with different structure are not exactly coincident with the dA₅-blocks and the B-DNA stretches. Within the dA₅ block, base-pair tilt and wedge roll adopt large values which proceed into the 3 flanking B-DNA sequence by about one base-pair. These properties of the structure calculated here are discussed in terms of different models explaining DNA curvature.     

Backbone and sidechain methyl Ile (δ1), Leu and Val chemical shift assignments of RDE-4 (1–243), an RNA interference initiation protein in C. elegans

The RNAi pathway of several organisms requires presence of double stranded RNA binding proteins for functioning of Dicer in gene regulation. In C. elegans, a double stranded RNA binding protein, RDE-4 (385 aa, 44 kDa) recognizes long exogenous dsRNA and initiates the RNAi pathway. We have achieved complete backbone and stereospecific methyl sidechain Ile (δ1), Leu and Val chemical shifts of first 243 amino acids of RDE-4, namely RDE-4ΔC.     

Interaction of Adenosine 5′-Triphosphate with Metal Ions X-ray Structure of Ternary Complexes Containing Mg(II), Ca(II), Mn(II), Co(II), ATP and 2,2′ -Dipyridylamine

Abstract

The X-ray structures of the isomorphous Mg²⁺, Ca²⁺, Mn²⁺ and Co²⁺ complexes of ATP have been determined. The metal ions are wrapped in hexa-coordination by the α, β and γ phosphate groups of two ATP molecules thus blocking the interaction of the metal ions with the adenine base. A second metal ion which is fully hydrated, M(H₂O)²⁺ ₆, is engaged in a strong hydrogen bond with the γ phosphate group of ATP and suggests a possible step in facilitating the cleavage between the β and γ phosphates in phosphoryl transfer reactions.