Aqueous plant extracts as stimulators of laccase production in liquid cultures of Lentinus edodes

Summary Total and specific activities of extra-cellular laccases from Lentinus edodes were enhanced by adding corn straw and chestnut juice to the liquid growth medium. The aqueous extracts were chemically characterized and revealed the presence of several phenolic and non-phenolic compounds. Extensive extraction of these components from the tested extracts completely annulled their stimulating properties on laccase production, suggesting that these compounds can act at micromole levels.     

A sensitive RNase protection assay using33P labeled antisense riboprobes

This article presents for the first time a modified protocol for RNase protection analysis that allows the substitution of³²P with³³P without loss of the high sensitivity of this method achieved with³²P. With this protocol, we were able to detect at least 1 pg of specific mRNA. In the RNase protection analysis³³P labeled riboprobes are more advantageous with regard to an easier handling and better resolution.     

The K/Na and Ca/Na ratios and rapeseed yield,under soil salinity or sodicity

Rapeseed (Brassica napus) is a crop relatively tolerant to salt and sodium. Our objective was to study the interactions between Na, K and Ca and their relationship with its yield under the isolated effects of soil salinity or sodicity.Two experiments were carried out using pots filled with the Ah horizon of a Typic Natraquoll. There were three salinity levels (2.3 dS m^-1; 6.0 dS m^-1 and 10.0 dS m^-1) and three sodicity levels, expressed as sodium adsorption ratios (SAR: 12; 27 and 44). The soil was kept near field capacity.As soil salinity increased, the K/Na and Ca/Na ratios in the tissues decreased markedly but yields and aerial biomass production were not affected. As soil SAR value increased, the K/Na and Ca/Na ratios in plants and K-Na and Ca-Na selectivities decreased. Plants could not maintain their Ca concentration in soil with a high SAR. The grain yield and biomass production diminished significantly in the highest SAR treatment. Our results are consistent with those showing detrimental osmotic effects of salts in Brassica napus. Conversely, under sodicity, the K/Na and Ca/Na ratios in plant tissues decreased considerably, in accordance with grain and biomass production. These results show that the effects of sodicity are different from those of salinity.     

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

Reactions of the haloalcohols HO(CH2)nCl (n = 2, 3) with platinum(II) - alkynyl and -alkyne complexes. X-ray crystal structure of trans-[Pt(Me) {C(OCH2CH2Cl) (CH2Ph)} (PPh3)2] [BF4]

The chloro complexes trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄, react with 1-alkynes HC---C---R in the presence of NEt₃ to afford the corresponding acetylide derivatives trans-[Pt(Me) (C---C---R) (PPh₃)₂] (R = p-tolyl (1), Ph (2), C(CH₃)₃ (3)). These complexes, with the exception of the t-butylacetylide complex, react with the chloroalcohols HO(CH₂)_nCl (n = 2, 3) in the presence of 1 equiv. of HBF₄ to afford the alkyl(chloroalkoxy)carbene complexes trans-[Pt(Me) {C[O(CH₂)_nCl](CH₂R) } (PPh₃)₂][BF₄] (R = p-tolyl, N = 2 (4), N = 3 (5); R=Ph, N = 2 (6)). A similar reaction of the bis(acetylide) complex trans-[Pt(C---C---Ph)₂(PMe₂Ph)₂] with 2 equiv. HBF₄ and 3-chloro-1-propanol affords trans-[Pt(C---CPh) {C(OCH₂CH₂CH₂Cl)(CH₂Ph) } (PMe₂Ph)₂][BF₄] (7). T alkyl(chloroalkoxy)-carbene complex trans-[Pt(Me) {C(OCH₂CH₂Cl)(CH₂Ph) } (PPh₃)₂][BF₄] (8) is formed by reaction of trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄ in HOCH₂CH₂Cl, with phenylacetylene in the presence of 1 equiv. of n-BuLi. The reaction of the dimer [Pt(Cl)(μ-Cl)(PMe₂Ph)]₂ with p-tolylacetylene and 3-chloro-1-propanol yields cis-[PtCl₂{C(OCH₂CH₂CH₂Cl)(CH₂C₆H₄-p-Me}(PMe₂Ph)] (9). The X-ray molecular structure of (8) has been determined. It crystallizes in the orthorhombic system, space group Pna2₁, with a = 11.785(2), B = 29.418(4), C = 15.409(3) Å, V = 4889(1) ų and Z = 4. The carbene ligand is perpendicular to the Pt(II) coordination plane; the PtC(carbene) bond distance is 2.01(1) Å and the short C(carbene)-O bond distance of 1.30(1) Å suggests extensive electronic delocalization within the Pt---C(carbene)---O moietry.     

Pro-Thyrotropin-Releasing Hormone Processing by Recombinant PC1

Abstract: Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH₂), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [³H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH_25–152 or preproTRH_25–158) and a 10-kDa C-terminal peptide (preproTRH_154–255 or preproTRH_160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (preproTRH_25–76 or preproTRH_25–82) and a 3.8-kDa peptide (preproTRH_83–108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (preproTRH_206–255). The optimal pH for these cleavages was 5.5. ZnCl₂, EDTA, EGTA, and the omission of Ca²⁺ inhibited the formation of pYE₂₇ (preproTRH_25–50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

Analysis of wheat resistance to the cereal aphidSitobion avenae using electrical penetration graphs and flow charts combined with correspondence analysis

The behaviour ofSitobion avenae (F.), was compared on resistant wheat lines ofTriticum monococcum (L.) and a susceptible variety ofTriticum aestivum (L.). Firstly, stylet penetration activities were monitored with the Electrical Penetration Graph (EPG) technique and subsequently analysed using flow charts combined with correspondence analysis. Plant resistance was shown to be associated with repeated penetrations without access to either the xylem or the phloem, and with numerous failures in starting a sustained sap ingestion (as represented by pattern E2). Access to sieve elements of the phloem did not seem to be much affected on resistant plants but it took the aphid three times as long to produce a sap ingestion pattern when maintained on the resistant lineT. monococcum n^o 44 (Tm44) as compared with aphids maintained on susceptible plants. As a result the total time spent in ingesting from sieve elements was reduced by 72% on Tm44. Secondly, direct observations of freely-moving apterous adults were performed. Aphids did not discriminate between resistant and susceptible wheat during the first 30 min of access to test leaves, but only 4 out of 25 aphids were still probing after eight hours on resistant Tm44. The relevance of these results to possible location of the resistance factor(s) are discussed. Although detection of plant resistance before sieve elements are reached can not be rigorously excluded, the factors involved inT. monococcum resistance toS. avenae undoubtedly occur within the phloem vessels.     

Cell-contact mediated modulation of the sialylation of contactinhibin

Contactinhibin was found to be involved in contact-dependent inhibition of growth. The growth inhibitory activity of contactinhibin is mediated by N-linked oligosaccharides with desialylated -glycosidically linked, terminal galactose residues. Here we show that in sparse human fibroblasts contactinhibin was expressed in a biologically inactive, highly sialylated form both on the plasma membrane and intracellularily, while in confluent cells plasma membrane localized contactinhibin was present in a biologically active, low sialylated form. Plasma membranes were shown to contain a glycoprotein sialidase which is suggested to be engaged in the activation of contactinhibin in a cell contact-dependent manner.