Silva Fennica Vol. 14, 1980.

Julkaistu Silva Fennica Vol. 14(4) -numeron liitteenä.

The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) angstrom and a = 6.023(3) angstrom, b = 11.719(3) angstrom and b = 13.455(4) angstrom, c = 20.409(3) angstrom and c = 20.702(4) angstrom for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.

Annales Societatis zoologicae-botanicae Fennicae Vanamo tom. 14

Kielet saksa ja suomi.

Structure of pregna-4,16-diene-7[alpha],14[alpha]-diol-3,20-dione

7a,14a-Dihydroxypregna-4,16-diene-3,20- dione, C21H2804, M r = 344.45, orthorhombic, P212121, a = 7.136 (1), b = 12.342 (1), c = 20.049 (3)/k, V= 1765.7 (3)/k 3, Z = 4, Dx = 1.295 g cm -3, A(Cu Kte) = 1.5418/k, /z = 6.7 cm- a, F(000) = 744, T = 293 K, R = 0.048 for 1345 observations. The A ring may be described as in a l a,2flhalf- chair conformation or a l a-sofa conformation. The B and C rings adopt normal chair conformations and the D ring has a 14a-envelope conforma tion. The molecules are held together by a hydrogen bond [0(3)...0(7)= 2.767 A].

Excitation and correlation of N-14 overtone transitions and measurement of heteronuclear dipolar coupling using DAPT

For studying systems containing nitrogen, limited use of N-14 NMR spectroscopy has been made because of the large quadrupolar interaction experienced by the N-14 nucleus and the absence of a central transition. To overcome the above problem, use of overtone spectroscopy has been suggested. Though this approach has limited applicability for powder samples due to second order quadrupole broadening, it is useful for studying oriented samples and single crystals. Here, we demonstrate the use of the recently proposed dipolar assisted polarization transfer (DAPT) pulse scheme for exciting the overtone transitions. The pulse sequence may also be utilized as a two-dimensional experiment to obtain H-1-N-14 dipolar couplings and H-1 chemical shifts. (C) 2010 Elsevier B.V. All rights reserved.

Studies on the 14α-hydroxylation of progesterone in mucor piriformis

Cell-free extracts with high 14?-hydroxylase activity were prepared from induced vegetative cell cultures of Mucor piriformis by grinding in potassium phosphate buffer (0.05 M, pH 8.0) containing glucose (0.25 M), KCl (1 mM), glutathione (1.0 mM) and glycerol (10%). Although the ideal pH for preparing the cell-free extract from vegetative cells was 8.0, the pH optimum of the hydroxylase was found to be 7.6. Microsomes (2.0 mg) prepared from the crude cell-free extract hydroxylated progesterone to 14?-hydroxyprogesterone in not, vert, similar60% yields in 30 min in the presence of NADPH and O2. Microsomes prepared from the uninduced cells did not contain any 14?-hydroxylase activity. The hydroxylase activity was inhibited to a significant extent by CO and p-chloromercuribenzoate whereas moderate inhibition was noticed in the presence of SKF-525A, metyrapone and N-methylmaleimideindicating the possible involvement of the cytochromeP-450 system in the reaction. The membrane bound hydroxylase was solubilized using Triton X-100 and the solubilized fraction contained nearly 35% of the original hydroxylase activity.

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.

Characterization of a 10- to 14-kilodalton protease-sensitive mycobacterium tuberculosis H37Ra antigen that stimulates human -yi T cells

gamma delta T-cell receptor-bearing T cells (gamma delta T cells) are readily activated by intracellular bacterial pathogens such as Mycobacterium tuberculosis. The bacterial antigens responsible for gamma delta T-cell activation remain poorly characterized. We have found that heat treatment of live M. tuberculosis bacilli released into the supernatant an antigen which stimulated human gamma delta T cells, gamma delta T-cell activation was measured by determining the increase in percentage of gamma delta T cells by flow cytometry in peripheral blood mononuclear cells stimulated with antigen and by proliferation of gamma delta T-cell lines with monocytes as antigen-presenting cells. Supernatant from heat-treated M. tuberculosis was fractionated by fast-performance liquid chromatography (FPLC) on a Superose 12 column. Maximal gamma delta T-cell activation was measured for a fraction of 10 to 14 kDa. Separation of the supernatant by preparative isoelectric focusing demonstrated peak activity at a pi of <4.0. On two-dimensional gel electrophoresis, the 10- to 14-kDa FPLC fraction contained at least seven distinct molecules, of which two had a pi of <4.5. Protease treatment reduced the bioactivity of the 10- to 14-kDa FPLC fraction for both resting and activated gamma delta T cells. Murine antibodies raised to the 10- to 14-kDa fraction reacted by enzyme-linked immunosorbent assay with antigens of 10 to 14 kDa in lysate of M. tuberculosis. In addition, gamma delta T cells proliferated in response to an antigen of 10 to 14 kDa present in M. tuberculosis lysate. gamma delta T-cell-stimulating antigen was not found in culture filtrate of M. tuberculosis but was associated,vith the bacterial pellet and lysate of M. tuberculosis. These results provide a preliminary characterization of a 10- to 14-kDa, cell-associated, heat-stable, low-pI protein antigen of M. tuberculosis which is a major stimulus for human gamma delta T cells.

IN-VITRO CYTOTOXICITY AND CELL CYCLE ANALYSIS OF TWO NOVEL BIS-1, 2, 4-TRIAZOLE DERIVATIVES: 1,4-BIS[5-(5-MERCAPTO-1,3,4-OXADIAZOL-2-yl-METHYL)-THIO-4-(p-TOLYL)-1, 2,4-TRIAZOL-3-yl]-BUTANE (MNP-14) AND 1,4-BIS[5-(CARBETHOXY-METHYL)-THIO-4-(p-ETHOXY PHENYL)-1,2,4-TRIAZOL-3-yl]-BUTANE (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio4-(p-tolyl)-1,2 ,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC50 of 3-5 mu M) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G1 phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg2+ and Mn2+ are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.

NMR Analysis of Cross Strand Aromatic Interactions in an 8 Residue Hairpin and a 14 Residue Three Stranded beta-Sheet Peptide

Cross strand aromatic interactions between a facing pair of phenylalanine residues in antiparallel beta-sheet structures have been probed using two structurally defined model peptides. The octapeptide Boc-(LFVPPLFV)-P-D-P-L-OMe (peptide 1) favors the beta-hairpin conformation nucleated by the type II' beta-turn formed by the (D)Pro-(L)Pro segment, placing Phe2 and Phe7 side chains in proximity. Two centrally positioned (D)Pro-(L)Pro segments facilitate the three stranded beta-sheet formation in the 14 residue peptide Boc-LFV(D)P(L)PLFVA(D)P(L)PLFV-OMe (peptide 2) in which the Phe2/Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides 1 and 2 are established by the delineation of intramolecularly hydrogen bonded NH groups and by the observation of specific cross strand NOEs. The observation of ring current shifted aromatic protons is a diagnostic of close approach of the Phe2 and Phe7 side chains. Specific assignment of aromatic proton resonances using HSQC and HSQC-TOCSY methods allow an analysis of interproton NOEs between the spatially proximate aromatic rings. This approach facilitates specific assignments in systems containing multiple aromatic rings in spectra at natural abundance. Evidence is presented for a dynamic process which invokes a correlated conformational change about the C-alpha-C-beta(chi(1)) bond for the pair of interacting Phe residues. NMR results suggest that aromatic ring orientations observed in crystals are maintained in solution. Anomalous temperature dependence of ring current induced proton chemical shifts suggests that solvophobic effects may facilitate aromatic ring clustering in apolar solvents.

The effect of minor addition of Ni on the microstructural evolution and mechanical properties of suction cast Al-0.14 at. pct Sc binary alloy

Aluminum scandium binary alloys represent a promising precipitation-hardening alloy system. However, the hardness of the binary alloys decreases with the rapid coarsening of Al3Sc precipitate during high-temperature aging. In the current study, we report a new approach to compensate for the loss of mechanical properties by combining rapid solidification with very small ternary addition of transition metal Ni. This addition yields dispersion, and at a critical concentration improves the mechanical properties. We explore additions of a maximum of 0.06 at. pct of Nickel to a binary Al-0.14 at. pct Sc alloy, which yield nickel-rich dispersions. We report two kinds of biphasic dispersions containing AlNi2Sc/Al9Ni2 and alpha-Al/Al9Ni2 phase combinations. The maximum improvement in mechanical properties occurs with the addition of 0.045 at. pct Ni with a yield strength of 239 +/- A 7 MPa for an aging treatment at 583 K (310 A degrees C) for 15 hours. DOI: 10.1007/s11661-013-1624-z (C) The Minerals, Metals & Materials Society and ASM International 2013