1,11-diarylundecan-1-one and 4-aryltetralone neolignans from Virola sebifera

The seed of Virola sebifera contains besides the polyketide 1 - (2′,6′ - dihydroxyphenyl) - 11 - henylundecan - 1 - one, four neolignans: (2S, 3S, 4R) - 4 - hydroxy - 2,3 - dimethyl - 5,6 - methylenedioxy - 4 - piperonyl - 1 - tetralone and its 2-epimer, as well as (2R, 3R, 4S) - 4 - hydroxy - 6,7 - dimethoxy - 2,3 - dimethyl 4 - piperonyl - 1 - tetralone and its (2R, 3S, 4R) - dehydroxy analogue.     

Expression of Paenibacillus polymyxa β-1,3-1,4-glucanase in Streptomyces lydicus A01 improves its biocontrol effect against Botrytis cinerea

Streptomyces lydicus strain A01, which can produce natamycin and chitinase, has a significant inhibition effect on gray mold disease caused by Botrytis cinerea. However, it has no detectable glucanase activity. Strain A21 isolated from the snow covered high altitude area in Tibet, China, also has a high antagonistic activity against B. cinerea. It displayed an obvious halo on lichen polysaccharides plates by congo red staining, indicating a strong glucanase activity. A21 was identified as Paenibacillus polymyxa using 16S rDNA gene analysis and biochemical and physiological analysis. To obtain the synergistic antifungal effects of natamycin, chitinase, and glucanases on B. cinerea, this study transformed the β-1,3-1,4-glucanase gene from P. polymyxa A21 to S. lydicus A01. The engineered S. lydicus AG01 showed substantially high glucanase activity, and had similar natamycin production and chitinase activity as the wild-type strain A01. Compared to the wild-type strain A01, the antifungal effects of S. lydicus AG01 on B. cinerea, including inhibition of spore germination and mycelial growth, were highly improved. The improved biocontrol effect of S. lydicus AG01 is likely attributed to the heterologous expression of glucanase from P. polymyxa, which acted synergistically with natamycin and chitinase to increase the antifungal activity of the strain.     

Molecular analysis of the TGF-beta controlled gene expression program in chicken embryo dermal myofibroblasts

The myofibroblast is a mesenchymal cell characterized by synthesis of the extracellular matrix, plus contractile and secretory activities. Myofibroblasts participate in physiological tissue repair, but can also cause devastating fibrosis. They are present in the tumor stroma of carcinomas and contribute to tumor growth and spreading. As myofibroblasts derive from various cell types and appear in a variety of tissues, there is marked variability in their phenotype. As regulatory mechanisms of wound healing are likely conserved among vertebrates, detailed knowledge of these mechanisms in more distant species will help to distinguish general from specific phenomena. To provide this as yet missing comparison, we analyzed the impact of the chemical inhibition of TGF-beta signaling on gene expression in chicken embryo dermal myofibroblasts. We revealed genes previously reported in mammalian systems (e.g. SPON2, ASPN, COMP, LUM, HAS2, IL6, CXCL12, VEGFA) as well as novel TGF-beta dependent genes, among them PGF, VEGFC, PTN, FAM180A, FIBIN, ZIC1, ADCY2, RET, HHIP and DNER. Inhibition of TGF-beta signaling also induced multiple genes, including NPR3, AGTR2, MTUS1, SOD3 and NOV. We also analyzed the effects of long term inhibition, and found that it is not able to induce myofibroblast dedifferentiation.     

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K_i values range from 0.38 mM for 6-deoxy-d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K_i=1.8(±0.1) mM], one by d-galactose [K_i=164(±11) mM] and one by d-mannose [K_i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK_a1=5.55(±0.15), pK_a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Serine-inserting UAA suppression mediated by yeast tRNASer

The number of loci that give rise to serine-inserting UAA suppressors in the yeast Saccharomyces cerevisiae was determined by examining over 100 of the revertants that suppressed the two UAA markers his4-1176 and leu2-1: the his4-1176 marker is suppressed by serine-inserting but not by tyrosine- or leueine-inserting suppressors and the leu2-1 marker is suppressed by all UAA suppressors. The suppressors could be assigned to one or other of the four loci: SUP16 and SUP17. which were previously known to yield serine-inserting suppressors, and SUP19 and SUP22. The chromosomal map position of SUP19 suggested that it may be allelic to the previously reported suppressor SUP20, while the SUP22 suppressor has not been described. Representatives of all of the four suppressors were found to insert serine at the UAA site in iso-1-cytochrome c from suppressed cyc1-72 strains. The degree of suppression by the serine-inserting suppressors was SUP16 > SUP17 > SUP19 > SUP22. The efficiency of suppression of each of the four serine suppressors was increased by the chromosomal mutation sal and by the cytoplasmic determinant ψ⁺. Read-through of the synthetase gene of the RNA bacteriophage Qβ in a cell-free system was used to demonstrate that tRNA^Ser from SUP16, SUP17 and SUP19 strains can translate UAA codons. In contrast, tRNA^Ser or total tRNA from SUP22 strains had no suppressing activity. The results suggest that the three loci SUP16, SUP17 and SUP19 encode iso-accepting species of tRNA^Ser, and that the UAA suppression is mediated by mutationally altered tRNA molecules. The mechanism of SUP22 suppression remains unknown.     

Functional homogeneity of P-700 in cyclic and non-cyclic electron transport reactions in thylakoids

The photoinduced turnover of P-700 (the reaction center chlorophyll a of photosystem I) in higher plant thylakoids was examined at room temperature by observation of the kinetics and amplitude of the transmission signal at 700 nm. The concentration of P-700 functional in cyclic and non-cyclic electron transfer reactions was compared. For the cyclic reactions mediated by N-methylphenazonium-p-methosulfate, 2,3,5,6-tetramethylphenylenediamine, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethylphenylenediamine and non-cyclic reactions utilizing either methylviologen or NADP⁺ as acceptor, the illuminated steady-state concentration of P-700⁺ was shown to be similar. The data support the concept of a homogeneous pool of P-700 that is capable of interaction in both cyclic and non-cyclic electron transfer reactions and are consistent with previous data obtained in vivo.The amplitude and kinetics of the P-700 signal were found to be very dependent upon the composition of the reaction medium and differences were noted for turnover in the cyclic and non-cyclic reactions. Specifically, at white light saturation, the addition of low concentrations of divalent cations, such as Mg²⁺ or Ca²⁺, had no effect on the signal amplitude during the cyclic reactions, but, in confirmation of previous work, caused an attenuation of the signal amplitude during non-cyclic flow. At low light intensities, the divalent cations caused a similar reduction in redox level of P-700 in the steady-state during non-cyclic flow and also reduced the rate of P-700 photooxidation in the cyclic reactions. The concentration of divalent cation that reduced the signal amplitude of P-700⁺ during non-cyclic flow was compared with that required for the stimulation of the variable component of fluorescence, and it was shown to be similar with half maximal effects at 1 mM Mg²⁺. The observations confirm that divalent cations control non-cyclic electron transport by an activation of Photosystem II in addition to regulating the distribution of excitation energy between the two photosystems.     

14-3-3ζ/τ heterodimers regulate Slingshot activity in migrating keratinocytes

Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an α6β4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3ζ, we demonstrate that in keratinocytes 14-3-3ζ/τ heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3ζ acts as a dominant negative and inhibits the interaction between 14-3-3τ and SSH1. These results implicate 14-3-3ζ/τ heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.     

Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called ‘butyrolactone autoregulators’ which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R,6R)- and (2S,3S,6S)-enantiomers] were also produced from (±)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S,6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.     

Yeast UAA suppressors effective in psi+ strains: leucine-inserting suppressors.

We have previously reported the isolation and characterization of UAA suppressors from a haploid strain of yeast Saccharomyces cerevisiae containing the ψ⁺ non-Mendelian determinant which increases the efficiency of action of certain suppressors (Ono et al., 1979). Most of the suppressors caused the insertion of either tyrosine or serine. In contrast, the pattern of suppression of nutritional markers suggested that the rare suppressor, SUP26, inserted in an amino acid other than tyrosine or serine. In this investigation we report the characterization of additional suppressors, similar to SUP26, that were isolated on a medium lacking uracil and containing canavanine; this medium is expected to exclude serine-inserting suppressors because they do not suppress the ura4-1 marker, and to exclude tyrosine-inserting suppressors because they suppress the can1-100 marker. The total of 155 revertants similar to the SUP26 suppressor were analyzed genetically and these could be assigned to one or another of the six distinct loci SUP26, SUP27, SUP28, SUP29, SUP32 and SUP33. The SUP26, SUP27 and SUP29 loci mapped on chromosomes XII, IV and X, respectively. The detailed map position of the SUP29 suppressor suggests that it may be allelic to the SUP30 suppressor reported by Hawthorne &; Mortimer (1968). These six suppressors had the same pattern of suppression of UAA nutritional markers and all of them had a similar low efficiency of action on the iso-1-cytochrome c mutation cyc1-72. The efficiency of each of these suppressors was increased by a chromosomal allo-suppressor, sal. Each of the six suppressors caused the insertion of leucine in iso-1-cytochrome c at the UAA site of the cyc1-72 mutation. It is suggested that the gene products of these suppressors are redundant forms of the same leucine transfer RNA.     

Structures, redox behavior, antibacterial activity and correlation with electronic structure of the complexes of nickel triad with 3-(2-(alkylthio)phenylazo)-2,4-pentanedione

3-(2-(Alkylthio)phenylazo)-2,4-pentanedione (HL), an O, N, S donor ligand, is used for the synthesis of Ni(II), Pd(II) and Pt(II) complexes. The spectroscopic (IR, UV-Vis, and NMR) data determine the structure. The single crystal X-ray diffraction measurement of [Ni(L)₂] and [Pt(L)Cl] has confirmed the structures. Coulometric oxidation of [Ni(L)₂] and EPR spectra thereof show formation of Ni(III) state. DFT computation has calculated the electronic configuration and has explained the spectral and redox properties of the complexes. The compounds are screened for their in vitro anti-bacterial activity using Gram-positive and Gram-negative bacteria (Bacillus subtilis UC564, Escherichia coli TG1, Staphylococcus aureus Bang25, Pseudomonas aeruginosa C/1/7, Salmonella typhi NCTC62, Salmonella paratyphi NCTC A2, Shigella dysenteriae 8NCTC599/52, Streptococcus faecalis S2, Vibrio cholerae DN7 and Mricococcus luteusAGD1). The minimum inhibitory concentration is determined for the compounds. The effect of the structure of the investigated compounds on the antibacterial activity is discussed.     

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in Complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with α-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, ?75 and 187 mV, respectively. In addition, two very small contributions to the α-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c₁ (Λ_m(25°C) = 553.5 nm; E′₀ = 238 mV) and four cytochromes bΛ_m(25°C) = 558.6, 561.2, 562.1, 566.1 nm and E′₀ = ?83, 26, 85, ?60 mV).     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Chlorophyll a forms in Phaeodactylum tricornutum: Comparison with other diatoms and brown algae

The long-wave chlorophyll a forms in Phaeodactylum tricornutum (688 and 703 nm) change into a short-wave form, 670 nm, as a result of incubation with 55% glycerol, freeze-thawing, short ultraviolet irradiation and, probably, chloroplast preparation. This short-wave form is non-fluorescent. Fluorescence polarisation measurements indicate that the long-wave chlorophyll a molecules are oriented parallel to each other. Although “labile” long-wave chlorophyll a receives energy from Photosystem II pigments at room temperatures and follows the induction phenomena of fluorescence, it is indicated by afterglow experiments that it probably does not participate in Photosystem II.Long-wave chlorophyll forms in Fucus are stable and probably are related to Photosystem I.     

Structure-activity dependence in some novel ring-substituted 3,3-dimethyl-1-phenyltriazenes. Genetic effects in Drosophila melanogaster and in Saccharomyces cerevisiae by a direct and a host-mediated assay

The structure-activity dependence of ten ring-substituted 3,3-dimethyl-1-phenyltriazenes (DMPT), 3,3-dimethyl-1-(3-pyridyl)-triazene (3-PyDMT) and of 3,3-dimethyl-1-(3-pyridyl-N-oxide)-triazene (3-PyODMT) was investigated by the induction of recessive lethal mutations in Drosophila melanogaster and of mitotic gene conversions in Saccharomyces cerevisiae using both direct and host-mediated assays. Significant differences in genetic effectiveness were detected not only between structurally related compounds but also between the responses of each test system to the same mutagen. Triazenes which are easily cleaved at physological conditions showed the highest genetic activity in the direct yeast test whereas stable triazenes, especially those with ortho and para positions blocked by a halogen, were most active in Drosophila. We have concluded that (1) the released arenediazonium cation is most probably responsible for the convertogenic activity in yeast; (2) metabolites, arising from hydroxylation of the methyl group, are essential for the mutagenic activity in Drosophila. A possible molecular basis which could account for the diversity in genetic effectiveness is discussed in terms of reaction mechanisms which can be predicted from the structural features of the tested triazenes.     

Protein phosphatases in developing Dictyostelium discoideum cells

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities (M_r 170 000 and 520 000) and one kemptide phosphatase activity (M_r 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.     

Molecular characterization of a 14-3-3 zeta gene from Plodia interpunctella: A potential marker for phylogenetic inference

The 14-3-3 proteins are a large family of approximately 30 kDa acidic proteins and acting in the regulation of many biological processes. In this study, a 14-3-3 zeta (Pi14-3-3z) gene from the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae) was isolated and characterized. The full-length cDNA of Pi14-3-3z is 1382 bp, including a 5'-untranslated region (UTR) of 141 bp, 3′-UTR of 497 bp and an open reading frame (ORF) of 744 bp encoding a polypeptide of 247 amino acids which contains a 14-3-3 homologues domain (PF00244). The deduced Pi14-3-3z protein sequence has 81%–100% identity with the homologues in comparison to with other individuals. qPCR analysis revealed that Pi14-3-3z was expressed at the four developmental stages and in all tissues tested. Based on the amino acid of 14-3-3z, phylogenetic analysis demonstrated a similar topology with the traditional classification, suggesting 14-3-3z protein has the potential value in phylogenetic inference.     

Cell association and degradation of α2-macroglobulin-trypsin complexes in hepatocytes and adipocytes

¹²⁵I-labelled α₂-macroglobulin-typrin complex (¹²⁵I-labelled α₂-macroglobulin·trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37°C. The association of 0.5 μg/ml ¹²⁵I-labelled α₂-macroglobulin·trypsin was inhibited by unlabelled α₂-macroglobulin·trypsin with a half-inhibition constant of about 8 μg/ml (11 nM). ¹²⁵I-Labelled α₂-macrioglubulin became cell-associated to a smaller extent (10–40% of that of α₂-macroglobulin·trypsin) and the half-inhibition constant was about 35 μg/ml in adipocytes. The cell associated of ¹²⁵I-labelled α-macroglobulin·trypsin was markedly inhibited by dansylcadaverin, bacitracin, omission of Ca²⁺ from the medium or pretreatment of the cell with trypsin. After incubation for 180 min more than 60% of the cell-associated ¹²⁵-Ilabelled α₂-macroglobulin·trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized marterial. ¹²⁵I-Labelled α₂-macroglobulin·trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 μg/ml ¹²⁵I-labelled α₂-macroglobulin was approx. 40% of that of ¹²⁵I-labelled α₂-macroglobulin·trypsin. Degradation of ¹²⁵I-labelled α₂-macroglobulin·trypsin was abolished by a high concentration (0.5 mg/ml) and α₂-macroglobulin·trypsin. It is concluded that α₂-macroglobulin·trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.