Thermospermine is not a minor polyamine in the plant kingdom

Thermospermine is a structural isomer of spermine, which is one of the polyamines studied extensively in the past, and is produced from spermidine by the action of thermospermine synthase encoded by a gene named ACAULIS5 (ACL5) in plants. According to recent genome sequencing analyses, ACL5-like genes are widely distributed throughout the plant kingdom. In Arabidopsis, ACL5 is expressed specifically during xylem formation from procambial cells to differentiating xylem vessels. Loss-of-function mutants of ACL5 display overproliferation of xylem vessels along with severe dwarfism, suggesting that thermospermine plays a role in the repression of xylem differentiation. Studies of suppressor mutants of acl5 that recover the wild-type phenotype in the absence of thermospermine suggest that thermospermine acts on the translation of specific mRNAs containing upstream open reading frames (uORFs). Thermospermine is a novel type of plant growth regulator and may also serve in the control of wood biomass production.     

Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.Bacillus thuringiensis subsp. israelensis has received considerable attention for mosquito control because of its specific and potent toxicity (15). B. thuringiensis subsp. israelensis-based microbial insecticides have been widely used as active components for integrated management of mosquitoes (11, 13, 33, 34). B. thuringiensis subsp. israelensis produces at least four major crystal toxins (Cry toxins), namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa (5). Cry4Aa exhibits specific toxicity against Anopheles, Aedes, and Culex mosquito larvae (15, 27). The 130-kDa Cry4Aa protoxin is released from the protein crystal upon ingestion by susceptible mosquito larvae and is activated by gut proteases into two protease-resistant fragments with molecular masses of 20 and 45 kDa through intramolecular cleavage of a 60-kDa intermediate (39). The three-dimensional structure of Cry4Aa has been determined by X-ray crystallography at a resolution of 2.8 Å (6). The structure of Cry4Aa is similar to the structures of previously characterized Cry toxins (24, 26, 31) that are composed of three domains (domains I, II, and III). In general, domain I, which is located in the N-terminal region, is composed of seven amphipathic α-helices and is thought to participate in membrane insertion. Domain II, which consists of three antiparallel β-sheets, is a putative receptor binding domain (Fig. ​(Fig.1).1). In particular, the loops in domain II that are exposed on the surface of the toxin molecule vary significantly in length and amino acid sequence among Cry toxins (31) and are thought to be receptor binding sites. Domain III in the C-terminal region contains two antiparallel β-sheets that form a β-sandwich fold with a jellyroll topology (31). Domain III is assumed to be involved in structural integrity, membrane protein recognition, or both (23, 24, 30).Open in a separate windowFIG. 1.Three-dimensional structure of Cry4Aa domain II. The structure was generated with PyMOL software (8) using the Cry4Aa PDB code (6). The amino acid sequences and corresponding regions of loops 1, 2, and 3 are indicated by blue, red, and yellow, respectively. The amino acid sequences of β-strands adjacent to the loops are underlined.The insecticidal mechanism of Cry toxin involves multiple steps, including ingestion by susceptible insects, solubilization in the alkaline midgut juice, activation by trypsin-like midgut proteases, binding to specific receptors on midgut epithelial cells, and then insertion into the plasma membrane followed by the formation of cation-selective channels or pores (26, 31, 34, 41). According to the colloid-osmotic lysis model, these channels or pores allow ions and water to pass into the cells, resulting in destruction of the membrane potential, cell swelling, cell lysis, and eventual death of the host (20, 21). Thus, the mechanism seems to be very complicated and is affected by multiple factors. The binding of the toxin to the specific receptor is considered a vital step for specific insecticidal activity (35). In fact, modification of the receptor molecules has been reported for insects resistant to certain Cry toxins (12, 22, 36).In a search for the functional structures of Cry4Aa, we previously constructed various loop replacement mutants with mutations in the three major loops in domain II and showed that the replacement of loop 2 resulted in a significant loss of mosquitocidal activity. Replacement of loops 1 and 3 of Cry4Aa also affected mosquitocidal activity, but it did not eliminate it (17). In this study, to further characterize the loops, we constructed Cry4Aa mutants in which individual amino acids in the loops were replaced with alanine and analyzed the mutants to determine their mosquitocidal activity against Culex pipiens larvae. We also analyzed the structural integrity of the Cry4Aa mutant proteins subjected to proteolytic digestion and their binding affinity to brush border membrane vesicles (BBMV) prepared from C. pipiens larvae.     

The Arf-like GTPase Arl8 Mediates Delivery of Endocytosed Macromolecules to Lysosomes in Caenorhabditis elegans

Late endocytic organelles including lysosomes are highly dynamic acidic organelles. Late endosomes and lysosomes directly fuse for content mixing to form hybrid organelles, from which lysosomes are reformed. It is not fully understood how these processes are regulated and maintained. Here we show that the Caenorhabditis elegans ARL-8 GTPase is localized primarily to lysosomes and involved in late endosome-lysosome fusion in the macrophage-like coelomocytes. Loss of arl-8 results in an increase in the number of late endosomal/lysosomal compartments, which are smaller than wild type. In arl-8 mutants, late endosomal compartments containing endocytosed macromolecules fail to fuse with lysosomal compartments enriched in the aspartic protease ASP-1. Furthermore, loss of arl-8 strongly suppresses formation of enlarged late endosome-lysosome hybrid organelles caused by mutations of cup-5, which is the orthologue of human mucolipin-1. These findings suggest that ARL-8 mediates delivery of endocytosed macromolecules to lysosomes by facilitating late endosome-lysosome fusion.     

Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A

RNA helicases are molecular motors that are involved in virtually all aspects of RNA metabolism. Eukaryotic initiation factor (eIF) 4A is the prototypical member of the DEAD-box family of RNA helicases. It is thought to use energy from ATP hydrolysis to unwind mRNA structure and, in conjunction with other translation factors, it prepares mRNA templates for ribosome recruitment during translation initiation. In screening marine extracts for new eukaryotic translation initiation inhibitors, we identified the natural product hippuristanol. We show here that this compound is a selective and potent inhibitor of eIF4A RNA-binding activity that can be used to distinguish between eIF4A-dependent and -independent modes of translation initiation in vitro and in vivo. We also show that poliovirus replication is delayed when infected cells are exposed to hippuristanol. Our study demonstrates the feasibility of selectively targeting members of the DEAD-box helicase family with small-molecule inhibitors.     

In vivo behavior of muscle fascicles and tendinous tissues of human gastrocnemius and soleus muscles during twitch contraction.

The present study investigated the differences between the human medial gastrocnemius (MG) and soleus (SOL) muscles in length changes of muscle fascicles and tendinous tissues during twitch contraction induced by an electrical nerve stimulus. Also, the time-course characteristics of twitch torque were related with changes in the length of muscle fascicles and tendinous tissues. No significant difference was observed between MG and SOL in contraction and half relaxation times of the changes in lengths and velocities of both muscle fascicles and tendinous tissues. The time-course of changes in twitch torque was nearly identical to that of the length of muscle fascicles and tendinous tissues. It was suggested that the behavior of MG and SOL during twitch contraction is practically similar in spite of their known physiological and architectural differences, and that the time-course of twitch torque is greatly influenced by the changes in the length of muscle fascicles and tendinous tissues.     

Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.     

Expression of a Soybean (Glycine max [L.] Merr.) Seed Storage Protein Gene in Transgenic Arabidopsis thaliana and Its Response to Nutritional Stress and to Abscisic Acid Mutations

Among the three subunits of [beta]-conglycinin, the 7S seed storage protein of soybean (Glycine max [L.] Merr.), expression of the [beta] subunit gene is unique. Accumulation of the [beta] subunit is enhanced in sulfate-deficient soybean plants, and its mRNA levels increase when abscisic acid (ABA) is added to the in vitro cotyledon culture medium. Transgenic Arabidopsis thaliana lines carrying a gene encoding the [beta] subunit was constructed and grown under sulfate deficiency. Accumulation of both [beta] subunit mRNA and protein were enhanced in developing A. thaliana seeds. Accumulation of one of the A. thaliana seed storage protein mRNAs was also enhanced by sulfate deficiency, although the response was weaker than that observed for the soybean [beta] subunit mRNA. When the aba1-1 or abi3-1 mutations were crossed into the transgenic A. thaliana line, accumulation of the [beta] subunit was significantly reduced, whereas accumulation of the A. thaliana seed storage protein was not greatly affected. These results indicate that soybean and A. thaliana share a common mechanism for response to sulfate deficiency and to ABA, although the sensitivity is different between the species. The transgenic A. thaliana carrying the [beta] subunit gene of [beta]-conglycinin will be a good system to analyze these responses.     

Isolation of an Arabidopsis thaliana Mutant,mto1, That Overaccumulates Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation)

We isolated Arabidopsis thaliana mutants that are resistant to ethionine, a toxic analog of methionine (Met). One of the mutants was analyzed further, and it accumulated 10- to 40-fold more soluble Met than the wild type in the aerial parts during the vegetative growth period. When the mutant plants started to flower, however, the soluble Met content in the rosette region decreased to the wild-type level, whereas that in the inflorescence apex region and in immature fruits was 5- to 8-fold higher than the wild type. These results indicate that the concentration of soluble Met is temporally and spatially regulated and suggest that soluble Met is translocated to sink organs after the onset of reproductive growth. The causal mutation, designated mto1, was a single, nuclear, semidominant mutation and mapped to chromosome 3. Accumulation profiles of soluble amino acids suggested that the mutation affects a later step(s) in the Met biosynthesis pathway. Ethylene production of the mutants was only 40% higher than the wild-type plants, indicating that ethylene production is tightly regulated at a step after Met synthesis. This mutant will be useful in studying the translocation of amino acids, as well as regulation of Met biosynthesis and other metabolic pathways related to Met.     

Protein Phosphorylation in the Sieve Tubes of Rice Plants

Proteins and their phosphorylation were examined in rice phloemsap that had been collected by the insect laser technique. Analysisby SDS-PAGE indicated that rice phloem sap contained over 150proteins. The total protein concentration was 150–200ng µl^-1. Analysis of proteins extracted from leaves, rootsand seeds revealed that several major low-molecular-weight proteinswere confined to the rice phloem sap. Maintenance of rice plantsunder stable environmental conditions was associated with aconstant complement of proteins in the phloem sap. Phosphorylation and dephosphorylation of proteins were detectedin the sieve tubes and occurred in a light dependent mannerwhen [     

Effects of sulfur nutrition on expression of the soybean seed storage protein genes in transgenic petunia

The 7S seed storage protein (β-conglycinin) of soybean (Glycine max [L]. Merr.) has three major subunits; α, α′, and β. Accumulation of the β-subunit, but not the α- and α′-subunits, has been shown to be repressed by exogenously applied methionine to the immature cotyledon culture system (LP Holowach, JF Thompson, JT Madison [1984] Plant Physiol 74: 576-583) and to be enhanced under sulfate deficiency in soybean plants (KR Gayler, GE Sykes [1985] Plant Physiol 78: 582-585). Transgenic petunia (Petunia hybrida) harboring either the α′- or β-subunit gene were constructed to test whether the patterns of differential expression were retained in petunia. Petunia regulates these genes in a similar way as soybean in response to sulfur nutritional stimuli, i.e. (a) expression of the β-subunit gene is repressed by exogenous methionine in in vitro cultured seeds, whereas the α′-subunit gene expression is not affected; and (b) accumulation of the β-subunit is enhanced by sulfur deficiency. The pattern of accumulation of major seed storage protein of petunia was not affected by these treatments. These results indicate that this mechanism of gene regulation in response to sulfur nutrition is conserved in petunia even though it is not used to regulate its own major seed storage proteins.