Phospholipase Cδ3 is a novel binding partner of myosin VI and functions as anchoring of myosin VI on plasma membrane

Phospholipase Cδ3 (PLCδ3) is a key enzyme in phosphoinositide metabolism, however, its physiological function remains unknown. Here we identified the Myosin VI (Myo6) as a binding partner of the PLCδ3. A tail region containing IQ motif and the cargo-binding domain of Myo6, and the C2 domain and PH domain of PLCδ3 were responsible sites for the interaction. Since Myo6 has been well analyzed as one of the "deafness genes" in mouse and human, we examined the expression pattern of PLCδ3 mRNA in the inner ear. In situ hybridization analysis indicated that both Myo6 and PLCδ3 were clearly and limitedly co-expressed in the inner and outer hair cells in the cochlea. Although actin structure of the stereocilia of hair cells seemed to be normal and no detectable hearing defect was observed in PLCδ3 knockout (KO) mice, stable PLCδ3 knockdown in Caco-2 colonic carcinoma cells caused abnormal actin structure of microvilli. In addition, dramatic decrease in expression of Myo6 was observed in intestine of PLCδ3KO mice, where microvilli structure is well developed. These results indicate that PLCδ3 could participate in stability of microvilli structure via regulating and anchoring of Myo6 to plasma membrane.     

Structural modeling of RNase P RNA of the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5′ leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure–function relationships of this multifaceted primordial ribozyme.     

中国假尾孢属的研究VI.

本文报道中国假尾孢属的16个种,其中有3个新种:藤山柳假尾孢(Pseudocercospora clematoclethrae),仙茅假尾孢(P. curculiginis,),豆付柴假尾孢(P. premnicola), 8个新组合:金粟兰假尾孢(P. chloranthe), 榅桲假尾孢(P. cydoniae),福岗假尾孢(P. fukuokaensis),土连翘假尾孢(P. hymenodictyonis),南五味子假尾孢(P. kadsurae), 野岛假尾孢(P. nojimae).绣线菊假尾孢(P. spiraeicola),球形假尾孢(P. sphaeriiformis)和3个中国新纪录。文中对新种进行了描述并绘图。研究的标本保存在中国科学院微生物研究所真菌标本室(HMAS)。     

Interactions between the tomato spotted wilt virus movement protein and plant proteins showing homologies to myosin, kinesin and DnaJ-like chaperones

The non-structural protein encoded by the M RNA segment (NSm) of tomato spotted wilt virus (TSWV) has been implicated in cell-to-cell movement of nucleocapsids through modified plasmodesmata. Recently, DnaJ-like proteins from Nicotiana tabacum (tobacco) and Arabidopsis thaliana have been identified as NSm interacting host proteins, implying an involvement of molecular chaperones during systemic spread of the virus or other, presently unknown NSm-mediated virus functions. Examination of additional TSWV host plants and improvement of yeast two-hybrid interaction trap experiments led to the isolation of a DnaJ-like protein from Lycopersicon esculentum (tomato) and the identification of a protein from A. thaliana sharing some homologies with myosin and kinesin-like polypeptides. Sequence alignments of the tomato DnaJ-like protein unveiled the corresponding gene as an orthologue to the tobacco and A. thaliana DnaJ genes, substantiating that NSm interacting DnaJ-like polypeptides, identified from three different TSWV host species, apparently form a subgroup distinct from archetypical DnaJ chaperones. Increased levels of DnaJ-like proteins could be detected in TSWV systemically infected leaves and in plants exposed to heat shock, showing that the NSm interacting DnaJ-like chaperones are inducible upon biotic and abiotic stress. All together, the identification of DnaJ-like proteins and a protein resembling myosin and kinesin as NSm interacting plant proteins is in accordance with results accomplished for movement proteins from other plant attacking viruses showing an involvement of molecular chaperones and the cytoskeleton in at least intracellular trafficking.     

Effects of lipid kinase expression and cellular stimuli on phosphatidylinositol 5-phosphate levels in mammalian cell lines

Phosphatidylinositol 5-phosphate (PtdIns5P) is a relatively recently discovered inositol lipid whose metabolism and functions are not yet clearly understood. We have transfected cells with a number of enzymes that are potentially implicated in the synthesis or metabolism of PtdIns5P, or subjected cells to a variety of stimuli, and then measured cellular PtdIns5P levels by a specific mass assay. Stable or transient overexpression of Type IIalpha PtdInsP kinase, or transient overexpression of Type Ialpha or IIbeta PtdInsP kinases caused no significant change in cellular PtdIns5P levels. Similarly, subjecting cells to oxidative stress or EGF stimulation had no significant effect on PtdIns5P, but stimulation of HeLa cells with a phosphoinositide-specific PLC-coupled agonist, histamine, caused a 40% decrease within 1 min. Our data question the degree to which inositide kinases regulate PtdIns5P levels in cells, and we discuss the possibility that a significant part of both the synthesis and removal of this lipid may be regulated by phosphatases and possibly phospholipases.     

Identification and characterization of splicing variants of PLEKHA5 (Plekha5) during brain development

PLEKHA5 (pleckstrin homology domain-containing protein family A, member 5) belongs to the PLEKHA family (PLEKHA1-6); however, the properties of this protein remain poorly characterized. We have identified and characterized two forms of PLEKHA5 mRNA. The long form of PLEKHA5 (L-PLEKHA5) contains 32 exons, encodes 1282 amino acids, and is specifically expressed in the brain; the short form of PLEKHA5 (S-PLEKHA5) is generated by alternative splicing of L-PLEKHA5, contains 26 exons, encodes 1116 amino acids, and is ubiquitously expressed. Both forms of the protein contain putative Trp-Trp (WW) and pleckstrin homology (PH) domains and are located mainly in the cytosol. Developmental and age-dependent expression studies in the mouse brain have shown that Plekha5 is the most abundantly expressed protein at E13.5 with S-Plekha5 dominancy. L-Plekha5 levels increased gradually with the decrease in total Plekha5 levels; moreover, L-Plekha5 became the dominant protein at E17.5, maintaining its dominance throughout adulthood. Protein-lipid overlay assays have indicated that the PH domain of PLEKHA5 specifically interacts with PI3P, PI4P, PI5P, and PI(3,5)P2. These results suggest that the S- to L-conversion of PLEKHA5 (Plekha5) may play an important role in brain development through association with specific phosphoinositides.     

Tea contains potent inhibitors of tyrosine phosphatase PTP1B

Tea is widely consumed all over the world. Studies have demonstrated the role of tea in prevention and treatment of various chronic diseases including diabetes and obesity, but the underlying mechanism is unclear. PTP1B is a widely expressed tyrosine phosphatase which has been defined as a target for therapeutic drug development to treat diabetes and obesity. In screening for inhibitors of PTP1B, we found that aqueous extracts of teas exhibited potent PTP1B inhibitory effects with an IC50 value of 0.4–4 g dry tea leaves per liter of water. Black tea shows the strongest inhibition activities, followed by oolong and then by green tea. Biochemical fractionations demonstrated that the major effective components in tea corresponded to oxidized polyphenolic compounds. This was further verified by the fact that tea catechins became potent inhibitors of PTP1B upon oxidation catalyzed by tyrosinases. When applied to cultured cells, tea extracts induced tyrosine phosphorylation of cellular proteins. Our study suggests that some beneficial effects of tea may be attributed to the inhibition of PTP1B.     

Involvement of myosin in intracellular motility and cytomorphogenesis in Micrasterias

Myosin was detected on Western blots of Micrasterias denticulata extracts by use of antibodies from different sources. Inhibitors with different targets of the actomyosin system, such as the myosin ATPase-blockers N-ethylmaleimide (NEM) and 2,3-butanedione monoxime (BDM), or the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapine (ML7), had similar effects on intracellular motility during cell development in the green alga Micrasterias, thus pointing towards a participation of myosin in these processes. The drugs markedly altered the mode of postmitotic nuclear migration, slowed down cytoplasmic streaming, changed cell pattern development and prevented normal chloroplast distribution and spreading into the growing semicell. In addition, an increase and dilatations in ER cisternae and marked morphological changes of the Golgi system were observed by transmission electron microscopy after exposure of growing cells to BDM.Neither BDM nor ML7 exhibited any effect on the distribution or arrangement of the cortical F-actin network nor on the F-actin basket around the nucleus, characteristic of untreated growing Micrasterias cells (J Cell Sci 107 (1994) 1929). This is particularly interesting since BDM caused disintegration of the microtubule system co-localized to the F-actin cage during normal nuclear migration. Together with the fact that other microtubules not connected to the F-actin system remained uninfluenced by BDM, this observation is evidence of an integrative function of myosin between the cytoskeleton elements.     

Nucleotide Sequence, Genomic Organization, and Chromosomal Localization of Genes Encoding the Human NMDA Receptor Subunits NR3A and NR3B

The N-methyl-D-aspartate (NMDA) receptors are glutamate-regulated ion channels that are critically involved in important physiological and pathological functions of the mammalian central nervous system. We have identified and characterized the gene encoding the human NMDA receptor subunit NR3A (GRIN3A), as well as the gene (GRIN3B) encoding an entirely novel subunit that we named NR3B, as it is most closely related to NR3A (57.4% identity). GRIN3A localizes to chromosome 9q34, in the region 13-34, and consists of nine coding exons. The deduced protein contains 1115 amino acids and shows 92.7% identity to rat NR3A. GRIN3B localizes to chromosome 19p13.3 and contains, as does the mouse NR3B gene (Grin3b), eight coding exons. The deduced proteins of human and mouse NR3B contain 901 and 900 amino acid residues, respectively (81.6% identity). In situ hybridization shows a widespread distribution of Grin3b mRNA in the brain of the adult rat.     

Unusual P450 reactions in plant secondary metabolism

Plant cytochromes P450 (P450s) participate in a variety of biochemical pathways to produce a vast diversity of plant natural products. The number of P450 genes in plant genomes is estimated to be up to 1% of the total gene annotations of each plant species, implying that plants are huge sources for various P450-dependent reactions. Plant P450s catalyze a wide variety of monooxygenation/hydroxylation reactions in secondary metabolism, and some of them are involved in unusual reactions such as methylenedioxy-bridge formation, phenol coupling reactions, oxidative rearrangement of carbon skeletons, and oxidative C–C bond cleavage. Here, we summarize unusual P450 reactions in various plant secondary metabolisms, and describe their proposed reaction mechanisms.     

Effect of Pam3Cys induced protection on the therapeutic efficacy of miltefosine against experimental visceral leishmaniasis

Prophylactic potential of synthetic bacterial lipopeptide and a TLR2 agonist, Pam3Cys was first evaluated against experimental visceral leishmaniasis in rodent model. After establishing the potential its effect on therapeutic efficacy of miltefosine was also studied. Pam3Cys showed 74.64% inhibition in parasitic establishment when administered by ip route at a dose of 100 μg/animal spaced at two weeks, i.e. on day −7 and +7 of challenge with Leishmania donovani amastigotes. However, when aforesaid dose of Pam3Cys was given with sub-curative dose of miltefosine (2.5 mg/kg for 5 days) its efficacy enhanced from 49.80% to 92.25%. These findings revealed that this lipopeptide has potential protective efficacy which significantly enhanced the therapeutic efficacy of miltefosine used at low dose against Leishmania infection and warrants detailed investigations on its possible immunopotentiatory actions.     

苏云金芽胞杆菌杀虫晶体蛋白的结构与功能研究进展

苏云金芽胞杆菌（Bacillus thuringiensis）杀虫晶体蛋白的毒性肽由三个典型的结构域组成.结构域Ⅰ位于肽链的N端,为一组由6～7个两亲的α螺旋围绕着一个疏水的α螺旋形成的α螺旋束,参与了细胞膜的穿孔;结构域Ⅱ位于肽链的中间,为三组以“希腊钥匙”（Greek key）拓扑结构连接在一起的反平行的β折叠片层,其顶端的突环参与了毒素与受体蛋白的结合;位于C端的结构域Ⅲ是由两组反平行的β折叠片层组成的夹心结构,以β果酱卷(jelly roll)拓扑结构排列,可能能够防止蛋白酶对毒素分子的过度降解.     

Identification of an artificial peptide motif that binds and stabilizes reduced human DJ-1

Although the precise biochemical function of DJ-1 remains unclear, it has been found to exert cytoprotective activity against oxidative stress. Cys106 is central to this function since it has a distinctly low pK_a rendering it extremely susceptible for oxidation. This characteristic, however, also poses a severe hindrance to obtain reduced DJ-1 for in vitro investigation. We have developed an approach to produce recombinant human DJ-1 in its reduced form as a bona fide basis for exploring the redox capacities of the protein. We solved the crystal structure of this DJ-1 at 1.56 Å resolution, allowing us to capture Cys106 in the reduced state for the first time. The dimeric structure reveals one molecule of DJ-1 in its reduced state while the other exhibits the characteristics of a mono-oxygenated cysteine. Comparison with previous structures indicates the absence of redox dependent global conformational changes in DJ-1. The capture of reduced Cys106 is facilitated by stabilization within the putative active site achieved through a glutamate side chain. This side chain is provided by a crystallographic neighbor as part of a ‘Leu–Glu’ motif, which was added to the C-terminus of DJ-1. In the structure this motif binds DJ-1 in close proximity to Cys106 through extended hydrophilic and hydrophobic interactions depicting a distinct binding pocket, which can serve as a basis for compound development targeting DJ-1.     

Light activation and molecular-mass changes of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase of spinach and maize leaves

Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W.m^–2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100 W.m^–2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach (fivefold on average) than in maize (twofold), which shows higher dark activity. The NADH dependent activity does not change appreciably. Several substrate activators can simulate in vitro the light effect with recovery of latent NADPH-dependent activity of spinach enzyme, but they are almost inactive with maize enzyme. A mixture of activators has been devised to fully activate the spinach enzyme under most conditions. The NAD(P)-GAPDH protein can be resolved by rapid gel filtration (fast protein liquid chromatography) into three conformers which have different molecular masses according to the light conditions. Enzyme from darkened leaves or chloroplasts, or dichlorophenyl-1,1-dimethylurea-treated chloroplasts is mainly a 600-kDa regulatory form with low NADPH-dependent activity relative to NADH-activity. Enzyme from spinach leaves or chloroplasts during photosynthesis is mainly a 300-kDa oligomer, which along with the 600-kDa form also occurs in leaves of darkened maize. The conformer of illuminated maize leaves is mainly a 160-kDa species. Results are consistent with a model of NAD(P)-GAPDH freely interconvertible between protomers of the 160-kDa (or 300-kDa intermediate) form with high NADPH-activity, produced in the light by the action of thioredoxin and activating metabolites (spinach only), and a regulatory 600-kDa conformer with lower NADPH-activity produced in darkness or when photosynthesis is inhibited. This behavior is reminiscent of the in-vitro properties of purified enzyme; therefore, it seems unlikely that NAD(P)-GAPDH in the chloroplast is part of a stable multienzyme complex or is bound to membranes.Abbreviations AEM activator equilibrium mixture - Chl chlorophyll - DCMU dichlorophenyl 1,1-dimethylurea - DTT dithiothreitol - FPLC fast protein liquid chromatography - NAD(P)-GAPDH glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent - PAR photosynthetic active radiation - PGK phosphoglycerate kinase - Tricine N-tris(hydroxy-methyl) methyl-glycine This work was supported by grants from the Ministero dell'Università e della Ricerca Seientifica e Tecnologica (40%, years 1990 and 1991).     

Lipid function in plant cell polarity

The establishment and maintenance of cell polarity play pivotal roles during plant development. During the past five years, proteins that are required for different aspects of plant cell polarity have been identified. However, the functions of lipids and their interactions with proteins that mediate polarity remained largely unaddressed. Recent genetic studies have discovered cell and tissue polarity mutants that have defects in sterol composition, glycosylphosphatidylinositol-anchored proteins, glycosylphosphatidylinositol biosynthesis and phospholipid signalling. Analyses of the affected gene products have provided a first glance at the roles of lipids in cell polarity signalling, as well as in the trafficking and anchoring of polar proteins.     

Contrasting responses of sulphate and phosphate transport in barley (Hordeum vulgare L.) roots to protein-modifying reagents and inhibition of protein synthesis

The uptake of sulphate into roots of barley seedlings is highly sensitive to phenylglyoxal (PhG), an arginine-binding reagent. Uptake was inhibited by >80% by a 1-h pre-treatment of roots with 0.45 mol · m^–3 PhG. Inhibition was maximal in pre-treatment solutions buffered between pH 4.5 and 6.5. Phosphate uptake, measured simultaneously by double-labelling uptake solutions with ³²P and ³⁵S, was less susceptible to inhibition by PhG, particularly at pH <6.5, and was completely insensitive to the less permeant reagent p-hydroxyphenylglyoxal (OH-PhG) administered at 1 mol · m^–3 at pH at 5.0 or 8.2; sulphate uptake was inhibited in -S plants by 90% by OH-PhG-treatment. Root respiration in young root segments was unaffected by OH-PhG pre-treatment for 1 h and inhibited by only 17% after 90 min pre-treatment. The uptake of both ions was inhibited by the dithiol-specific reagent, phenylarsine oxide even after short exposures (0.5–5.0 min). Sulphate uptake was more severely inhibited than that of phosphate, but in both cases inhibition could be substantially reversed by 5 min washing of treated roots by 5 mol · m^–3 dithioerythritol. After longer pre-treatment (50 min) with phenylarsine oxide, inhibition of the ion fluxes was not relieved by washing with dithioerythritol. Inhibition of sulphate influx by PhG was completely reversed by washing the roots for 24 h with culture solution lacking the inhibitor. The reversal was dependent on protein synthesis; less than 20% recovery was seen in the presence of 50 mmol · m^–3 cycloheximide. Sulphate uptake declined rapidly when -S roots were treated with cycloheximide. In the same roots the phosphate influx was little affected, small significant inhibitions being seen only after 4 h of treatment. Respiration was depressed by only 20% in apical and by 31% in basal root segments by cycloheximide pre-treatment for 2 h. Similar rates of collapse of the sulphate uptake and insensitivity of phosphate uptake were seen when protein synthesis was inhibited by azetidine carboxylic acid, p-fluorophenylalanine and puromycin. Considering the effects of all of the protein-synthesis inhibitors together leads to the conclusion that the sulphate transporter itself, or some essential sub-component of the uptake system, turns over rapidly with a half-time of about 2.5 h. The turnover of the phosphate transporter is evidently much slower. The results are discussed in relation to strategies for identifying the transport proteins and to the regulation of transporter activity during nutrient stress.Abbreviations CAP chloramphenicol - CHM cycloheximide - DTE dithioerythritol - OH-PhG p-hydroxyphenylglyoxal - PhAsO phenylarsine - PhG phenylglyoxal Paper dedicated to the memory of the late Ken Treharne who did much to encourage this collaboration.D.T.C. gratefully acknowledges a fellowship provided by Le Ministére des Etrangers during his stay in Montpellier.     

Immunolocalization of myosin in intact and wounded cells of the green algaErnodesmis verticillata (Kützing) Børgesen

Using in-vivo assays at least two classes of ethylene-binding site are shown to exist in pea epicotyls. These classes appear to have identical affinities for ethylene with a K_D of 6 · 10^–11-8 · 10^–11 M; the fast associating class has high and appropriate affinities for physiologically active analogues of ethylene, and CO₂ is without effect upon bfinding. Experiments involving suppression of endogenous ethylene production demonstrate that previous work may have underestimated both the amount of binding sites present and the affinity of such sites for ethylene. The apparent inhibition of binding by silver appears to be the consequence of a stimulation of endogenous ethylene production. The possible role of the sites as ethylene receptors is discussed.Abbreviations AOA amino oxyacetic acid - K_D dissociation constant We are grateful to the Biotechnology Action Programme of the European Economic Communities for support for this work.