Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW_XL and G3000SW_XL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SW_XL and G4000SW_XL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl₂ and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type. Received: 29 October 1998 / Accepted: 10 December 1998     

Cross-linking of plasmalemmal cholesterol in lymphocytes induces capping, membrane shedding, and endocytosis through coated pits.

By use of a nicked and biotinylated perfringolysin O (BCtheta), which binds to cholesterol specifically, we studied consequences of cross-linking cholesterol in lymphocytes. When bound with BCtheta and then with labeled avidin or streptavidin, capping occurred in most cells within 30 min at 37 degrees C. It was inhibited by cytochalasin D or NaN3, but not by nocodazole. When BCtheta-cholesterol was capped, Thy-1 and transferrin receptor, a GPI-anchored protein and a transmembrane protein, respectively, remained evenly distributed. By fluorescence and electron microscopy, a cluster of small vesicles bound with BCtheta were observed in the cap. They were then shed in the medium or internalized through coated pits. The result indicates that cross-linking of cholesterol in lymphocytes induces capping, but does not affect distribution of membrane proteins, and that the capped cholesterol molecules are either shed as vesicles or endocytosed.     

A new allele, DNASE1*6, of human deoxyribonuclease I polymorphism encodes an Arg to Cys substitution responsible for its instability.

A new allele, DNASE1*6, of human deoxyribonuclease I (DNase I) has been discovered by isoelectric focusing: its gene product has the most cathodic pI of the six electrophoretic variants. Results of DNA sequencing, mismatched PCR-restriction fragment length polymorphism, and transient transfection of the variant construct showed that the mutant was caused by a C-T transition at nucleotide position 1826, resulting in an Arg to Cys substitution at amino acid position 185 of the mature enzyme. The variant isoenzyme, expressed in COS-7 cells, was more labile than the other types. Instability and an increase in the pI value of the variant suggest that a structural alteration, perhaps due to aberrant formation of a disulfide bond, could occur in the enzyme.     

High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression

The mechanism of the arginine-rich peptide-mediated cellular uptake is currently a controversial issue. Several factors, including the type of peptide, the nature of the cargo, and the linker between them, appear to affect uptake. One of the less studied factors, which may affect the uptake mechanism, is the effect of peptide density on the surface of the cargo. Here, we examined the mechanism of cellular uptake and intracellular trafficking of liposomes modified with different densities of the octaarginine (R8) peptide. Liposomes modified with a low R8 density were taken up mainly through clathrin-mediated endocytosis, leading to extensive lysosomal degradation, whereas those modified with a high R8 density were taken up mainly through macropinocytosis and were less subject to lysosomal degradation. Furthermore, the high density R8-liposomes were able to stimulate the macropinocytosis-mediated uptake of other particles. When plasmid DNA was condensed and encapsulated in R8-liposomes, the levels of gene expression were three orders of magnitude higher for the high density liposomes. The enhanced gene expression by the high density R8-liposomes was highly impaired by blocking uptake through macropinocytosis. The different extents of gene expression from different densities of the R8 peptide on the liposomes could be explained principally by the existence of an intracellular trafficking route, but not by the uptake amount, of internalized liposomes. These results show that the density of the R8 peptide on liposomes determines the uptake mechanism and that this is directly linked to intracellular trafficking, resulting in different levels of gene expression.     

Therapeutic effect of anti-macrophage inflammatory protein 2 antibody on influenza virus-induced pneumonia in mice

We investigated the effect of anti-macrophage inflammatory protein 2 immunoglobulin G (aMIP-2 IgG) on the progression of influenza virus-induced pneumonia in mice. When mice were infected with a mouse lung-adapted strain of influenza A/PR/8/34 virus by intranasal inoculation, neutrophil counts in the bronchoalveolar lavage fluid (BALF) increased in parallel with the kinetics of MIP-2 production, which peaked 2 days after infection. After intracutaneous injection of a dose of 10 or 100 microg of aMIP-2 IgG once a day on days 0 and 1, neutrophil counts in BALF on day 2 were reduced to 49 or 37%, respectively, of the value in the control infected mice administered anti-protein A IgG. The antibody administration also improved lung pathology without affecting virus replication. Furthermore, by prolonged administration with a higher or lower dose for up to 5 days, body weight loss became slower and finally 40% of mice in both treatment groups survived potentially lethal pneumonia. These findings suggest that MIP-2-mediated neutrophil infiltration during the early phase of infection might play an important role in lung pathology. Thus, MIP-2 was considered to be a novel target for intervention therapy in potentially lethal influenza virus pneumonia in mice.     

PANP is a novel O-glycosylated PILRα ligand expressed in neural tissues

Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.     

A Randomized,Single-Blind,Placebo-Controlled Study on the Efficacy of the Arthrokinematic Approach-Hakata Method in Patients with Chronic Nonspecific Low Back Pain

Study design

cized, single-blind, controlled trial.

Objective

To investigate the efficacy of the Arthrokinematic approach (AKA)-Hakata (H) method for chronic low back pain.

Summary of Background Data

The AKA-H method is used to manually treat abnormalities of intra-articular movement.

Methods

One hundred eighty-six patients with chronic nonspecific low back pain randomly received either the AKA-H method (AKA-H group) or the sham technique (S group) monthly for 6 months. Data were collected at baseline and once a month. Outcome measures were pain intensity (visual analogue scale [VAS]) and quality of life (the Roland-Morris Disability Questionnaire [RDQ] and Short Form SF-36 questionnaire [SF-36]).

Results

At baseline, the VAS, RDQ, and SF-36 scores showed similar levels between the groups. After 6 months, the AKA-H group had more improvement in the VAS (42.8% improvement) and RDQ score (31.1% improvement) than the sham group (VAS: 10.4% improvement; RDQ: 9.8% improvement; both, P < 0.001). The respective scores for the SF-36 subscales (physical functioning, role physical, bodily pain, social functioning, general health perception, role emotional, and mental health) were also significantly more improved in the AKA-H group than in the sham group (all, P < 0.001). The scores for the physical, psychological, and social aspects of the SF-36 subscales showed similar improvement in the AKA-H group.

Conclusion

The AKA-H method can be effective in managing chronic low back pain.

Trial Registration

UMIN Clinical Trials Registry (UMIN-CTR) UMIN000006250.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from Marine Environments in Tokyo Bay

Pseudomonas aeruginosa is a pathogenic bacterium that has been thoroughly investigated since the 19th century and is generally regarded as a freshwater or terrestrial organism. In 1995, it was reported that the OprP porin, an outer membrane protein corresponding to that of this bacterium, was widely distributed as a dissolved component in seawater. This finding led us to investigate the presence of P. aeruginosa in marine environments. Both culture-independent and -dependent methods were applied to seawater samples obtained in Tokyo Bay during four cruises. The DVC-FA (direct viable count–fluorescent antibody) technique showed that cells reactive to an antibody against P. aeruginosa were widely present in the bay, i.e., 10³ to 10⁴ cells/mL in the inner bay, and 10² to 10³ cells/mL at the mouth. Bacterial cells isolated by selective medium were identified by three methods: the presence of oprI and oprL, two outer membrane lipoprotein genes specific to P. aeruginosa; the API20 NE kit; and 16S rDNA sequence analysis. The results confirmed that the majority of isolates from the bay were P. aeruginosa. Immuno-chemical analyses of the seawater results indicate that P. aeruginosa is commonly present in coastal marine environments and sheds OprP.     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.