The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.     

Human neuroblastoma GOTO cells express CD44 and localize it into lipid rafts upon differentiation into Schwannian cells

Neuroblastoma, which is a malignant tumor consisting of dedifferentiated neuroectodermal cells, is known to show spontaneous maturation or regression in its growth. Cultured human neuroblastoma GOTO cells could be induced to differentiate into Schwannian cells and neuronal cells by incubation in the presence of 5-bromo-2'-deoxyuridine (BrdU) and by serum depletion, respectively. Here we report that in association with these differentiations, cells differentiated into Schwannian cells specifically expressed a cell adhesion molecule CD44, of which expression is usually suppressed in GOTO cells. In contrast, it remained suppressed in cells differentiated into neuronal cells. Polymerase-chain reaction revealed that the CD44 species expressed was the hemopoietic form (CD44H) with long cytoplasmic tail. Furthermore, the newly expressed CD44 in the cells was found exclusively in membrane microdomains, called lipid rafts. These data suggest that CD44 might play an important role in GOTO cells differentiated into Schwannian cells.     

Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass-associated missense mutations in LRP5 affect canonical Wnt signaling

The low-density-lipoprotein receptor-related protein 5 (LRP5), a coreceptor in the canonical Wnt signaling pathway, has been implicated in human disorders of low and high bone mass. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma syndrome, and heterozygous missense mutations in families segregating autosomal dominant high bone mass (HBM) phenotypes have been identified. We expressed seven different HBM-LRP5 missense mutations to delineate the mechanism by which they alter Wnt signaling. None of the mutations caused activation of the receptor in the absence of ligand. Each mutant receptor was able to reach the cell surface, albeit at differing amounts, and transduce exogenously supplied Wnt1 and Wnt3a signal. All HBM mutant proteins had reduced physical interaction with and reduced inhibition by DKK1. These data suggest that HBM mutant proteins can transit to the cell surface in sufficient quantity to transduce Wnt signal and that the likely mechanism for the HBM mutations' physiologic effects is via reduced affinity to and inhibition by DKK1.     

The proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for cleavage of von Willebrand factor

ADAMTS13 limits platelet-rich thrombosis by cleaving von Willebrand factor at the Tyr(1605)-Met(1606) bond. Previous studies showed that ADAMTS13 truncated after spacer domain remains proteolytically active or hyperactive. However, the relative contribution of each domain within the proximal carboxyl terminus of ADAMTS13 in substrate recognition and specificity is not known. We showed that a metalloprotease domain alone was unable to cleave the Tyr-Met bond of glutathione S-transferase (GST)-VWF73-H substrate in 3 h, but it did cleave the substrate at a site other than the Tyr-Met bond after 16-24 h of incubation. Remarkably, the addition of even one or several proximal carboxyl-terminal domains of ADAMTS13 restored substrate specificity. Full proteolytic activity, however, was not achieved until all of the proximal carboxyl-terminal domains were added. The addition of TSP1 2-8 repeats and two CUB domains did not further increase proteolytic activity. Furthermore, ADAMTS13 truncated after the spacer domain with or without metalloprotease domain bound GST-VWF73-H with a K(d) of approximately 7.0 or 13 nm, comparable with full-length ADAMTS13 (K(d) = 4.6 nm). Metalloprotease domain did not bind GST-VWF73-H detectably, but the disintegrin domain, first TSP1 repeat, Cys-rich domain, and spacer domain bound GST-VWF73-H with K(d) values of 489, 136, 121, and 108 nm, respectively. These proximal carboxyl-terminal domains dose-dependently inhibited cleavage of fluorescent resonance energy transfer (FRETS)-VWF73 by full-length ADAMTS13 and ADAMTS13 truncated after the spacer domain. These data demonstrated that the proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for recognition and cleavage of von Willebrand factor between amino acid residues Asp(1595) and Arg(1668).     

A part of ice nucleation protein exhibits the ice-binding ability

We generated a recombinant 96-residue polypeptide corresponding to a sequence Tyr176-Gly273 of ice nucleation protein from Pseudomonas syringae (denoted INP96). INP96 exhibited an ability to shape an ice crystal, whose morphology is highly similar to the hexagonal-bipyramid generally identified for antifreeze protein. INP96 also showed a non-linear, concentration-dependent retardation of ice growth. Additionally, circular dichroism and NMR measurements suggested a local structural construction in INP96, which undergoes irreversible thermal denaturation. These data imply that a part of INP constructs a unique structure so as to interact with the ice crystal surfaces.     

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.     

Regulation of high glucose-induced apoptosis by mitochondrial NADP+-dependent isocitrate dehydrogenase

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic kidney complications, and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidant stress. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPm activity in HEK293 cells, an embryonic kidney cell line, regulates high glucose-induced apoptosis. When we examined the protective role of IDPm against high glucose-induced apoptosis with HEK293 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPm plays an important protective role in apoptosis of HEK293 cells induced by a high concentration of glucose and may contribute to various pathologies associated with the long-term complications of diabetes.     

Cooperative catalysis in the homodimer subunits of xanthine oxidase

Xanthine oxidase (XOD) consists of two identical subunits. For the past 50 years or so, it was assumed that the two subunits carry out catalysis independently. Herein, we report that the presence of 6-formylpterin (6FP) or other substrates (such as xanthine or xanthopterin) at one of the two active sites affects the binding affinity and catalysis rate of 6FP at the other. When the two XOD active sites were occupied by two 6FPs simultaneously, the conversion rate (2.8 x 10(-3) s(-1)) of 6FP to 6CP is 2.95-fold faster than the conversion rate (0.95 x 10(-3) s(-1)) in the case of single 6FP bound condition. The presence of xanthine can accelerate the catalysis rate of 6FP by XOD as well as the activity-recovering rate of alloxanthine-inhibited XOD. Our experimental observations demonstrate unambiguously that the two XOD subunits are strongly cooperative in both binding and catalysis. The inhibition constant (Ki) of 6FP toward XOD was measured by a stopped-flow method to be 0.94 nM.     

Expression of cytochrome c oxidase subunit 1 gene in the brain at an early stage in the termination of pupal diapause in the sweet potato hornworm, Agrius convolvuli

Suppression-subtractive hybridization was used to isolate cDNAs specifically expressed in the brain at the termination of pupal diapause in Agriusconvolvuli. One of the isolated clones shows similarity to the cytochrome c oxidase subunit 1 (COX1) gene. The full-length cDNA was obtained from brain mRNA by rapid amplification of cDNA ends (RACE). The insert is 1.65 kb in length and has an open reading frame of 1.46 kb which encodes a putative protein of 486 amino acid residues. RT-PCR reveals that the mRNA increases dramatically at an early stage of diapause termination. Activity of cytochrome c oxidase in the brain also increases at the same time. The up-regulation of this gene suggests that expression of the COX1 gene and ATP synthesis are initiated in the brain in association with diapause termination.     

Synaptic scaffolding molecule interacts with axin

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N-methyl-d-aspartate receptor subunits, neuroligin, and beta-catenin. Here, we identified Axin as a novel binding partner of S-SCAM. Axin was co-immunoprecipitated with S-SCAM from rat brain, detected in the post-synaptic density fraction in rat brain subcellular fractionation, and partially co-localized with S-SCAM in neurons. The guanylate kinase domain of S-SCAM directly bound to the GSK3beta-binding region of Axin. S-SCAM formed a complex with beta-catenin and Axin, but competed with GSK3beta for Axin-binding. Thereby, S-SCAM inhibited the Axin-mediated phosphorylation of beta-catenin by GSK3beta.