Crystal structures of protein phosphatase-1 bound to motuporin and dihydromicrocystin-LA: elucidation of the mechanism of enzyme inhibition by cyanobacterial toxins

The microcystins and nodularins are tumour promoting hepatotoxins that are responsible for global adverse human health effects and wildlife fatalities in countries where drinking water supplies contain cyanobacteria. The toxins function by inhibiting broad specificity Ser/Thr protein phosphatases in the host cells, thereby disrupting signal transduction pathways. A previous crystal structure of a microcystin bound to the catalytic subunit of protein phosphatase-1 (PP-1c) showed distinct changes in the active site region when compared with protein phosphatase-1 structures bound to other toxins. We have elucidated the crystal structures of the cyanotoxins, motuporin (nodularin-V) and dihydromicrocystin-LA bound to human protein phosphatase-1c (gamma isoform). The atomic structures of these complexes reveal the structural basis for inhibition of protein phosphatases by these toxins. Comparisons of the structures of the cyanobacterial toxin:phosphatase complexes explain the biochemical mechanism by which microcystins but not nodularins permanently modify their protein phosphatase targets by covalent addition to an active site cysteine residue.     

GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP

The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.     

Development of recombinant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> producing virus-like particles of a fish nervous necrosis virus

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.     

Transformation of Δ<Superscript>4</Superscript>-3-ketosteroids by free and immobilized cells of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> actinobacterium

9α-Hydroxy derivatives were prepared from 11 steroids of androstane and pregnane series using Rhodococcus erythropolis VKPM Ac-1740 culture with 0.5–10 g/l substrate concentration in the reaction mixture. 9α-Monohydroxylation proceeded regardless of the substituent structure at C17. However, the structure of the steroid molecule influenced the time of complete conversion of the substrate and the yield of the transformation product. 9α-Hydroxy-androstenedione was obtained in 35 h in a yield of 85% when the maximum concentration of androstenedione (AD) was 10 g/l. 9α-Hydroxy-AD was also formed by the actinobacterium cells entrapped in poly(vinyl alcohol) cryogel beads. Nine successive transformation cycles were carried out using immobilized cells at 4.0 g/l concentration of AD in the medium. The yield of 9α-hydroxy-AD formed during six cycles (from two to eight with the duration of each cycle for 22–24 h) was 98%.     

The clues to species specificity of pollination among Brassicaceae

The Brassica S product of the pollen-signalling S-gene (SP11 for Specificity Pollen, or SCR for S locus Cysteine Rich) has recently been identified as a member of the family of PCPs (Pollen Coat Proteins), a group of small basic proteins. In female plants SRK (S Receptor Kinase) has been demonstrated, among several stigma-expressed S genes, to exclusively control self-pollen rejection. Genes and gene products that are highly related to the sporophytic SI system appear to take part in the control of several stages of pollen-stigma interactions in the Brassicaceae. These genes ensure, in particular, the species specificity of late pollen adhesion. Pollen capture itself is not species specific, since it depends essentially on lipophilic affinities between pollen coatings and waxy pellicles on the stigma. These interactions and their control are reviewed here. Received: 2 August 2000 / Revision accepted: 11 August 2000     

Absence of DICER in Monocytes and Its Regulation by HIV-1

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host''s miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.     

Finite-time Lyapunov exponents and metabolic control coefficients for threshold detection of stimulus–response curves

In biochemical networks transient dynamics plays a fundamental role, since the activation of signalling pathways is determined by thresholds encountered during the transition from an initial state (e.g. an initial concentration of a certain protein) to a steady-state. These thresholds can be defined in terms of the inflection points of the stimulus–response curves associated to the activation processes in the biochemical network. In the present work, we present a rigorous discussion as to the suitability of finite-time Lyapunov exponents and metabolic control coefficients for the detection of inflection points of stimulus–response curves with sigmoidal shape.     

BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

A specific immunological probe for the major myelin proteolipid. Confirmation of a deletion in DM-20

Major myelin proteolipid (MMPL, also called PLP) and DM-20 are the two major intrinsic membrane proteins of CNS myelin. A specific immunological probe was obtained for MMPL by raising antibodies against the synthetic tridecapeptide 117-129 of MMPL. Antibodies against this peptide reacted with the MMPL but did not cross react with DM-20, while both proteolipids had been shown previously to be recognized by antibodies directed against the C-terminal hexapeptide of MMPL. This is in accordance with previous findings showing that DM-20 differs only from MMPL by a deletion of residues 100-140 (+/- few units). Furthermore, this site-specific immunological probe also recognizes MMPL in its native form in oligodendrocytes in primary glial cell cultures.     

鄂尔多斯东胜地区不同生态功能区的土壤侵蚀敏感性

利用ETM影像数据和GIS技术,对鄂尔多斯东胜地区进行生态功能分区.以水土流失通用方程 (universal soil loss equation, USLE) 理论为基础,并依据土壤侵蚀敏感性驱动力的相似性和差异性,分析了降水、土壤质地、地形和地表覆盖因子对该区域不同生态功能分区土壤侵蚀与水土流失敏感性的影响.研究结果表明:典型草原丘陵区土壤侵蚀敏感性指数SSj为4.2,典型草原平原区为4.0,典型草原沙化区为4.6,荒漠草原区土壤侵蚀敏感性指数为5.0;4个生态功能区的土地沙漠化都属于土地沙漠化高度敏感区,其由高到低的排序为荒漠草原区 > 典型草原沙化区 = 典型草原平原区 > 典型草原丘陵区;就土壤侵蚀程度而言,典型草原平原区属于土壤侵蚀轻度敏感区,荒漠草原区、典型草原沙化区、典型草原丘陵区属于土壤侵蚀中度敏感区.将其土壤侵蚀化程度由高到低排序:荒漠草原区 > 典型草原沙化区 > 典型草原丘陵区 > 典型草原平原区;就土壤盐渍化而言,典型草原丘陵区属于土地盐渍化不敏感区,其它3个区属于土地盐渍化轻度敏感区.在综合评价其土壤侵蚀敏感性区域差异基础上,阐述了鄂尔多斯东胜地区水土流失敏感性区域土地利用格局的调整与优化取向.