Neuronal leucine-rich repeat protein 4 functions in hippocampus-dependent long-lasting memory

Neuronal leucine-rich repeat proteins (NLRRs) are type I transmembrane proteins and expressed in neuronal tissues, but their function remains unknown. Here, we describe the identification and characterization of a new member of the NLRR family, NLRR4, and its potential role in long-lasting memory. We generated NLRR4-deficient (NLRR4(-/-)) mice and found that they showed impaired memory retention. In hippocampus-dependent learning tasks, NLRR4(-/-) mice were able to learn and maintain the memories for one day but unable to retain the memories for four days after learning. In contrast, in a hippocampus-independent task, NLRR4(-/-) mice were able to retain the memory normally for at least seven days. These results suggest that NLRR4 plays a key role in hippocampus-dependent long-lasting memory.     

Stable Transfection of GM1 Synthase Gene into GM1-Deficient NG108-15 Cells,CR-72 Cells,Rescues the Responsiveness of TRK-Neurotrophin Receptor to Its Ligand,NGF

Previous studies from this laboratory and others have suggested the evidences that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. The present study was aimed to examine whether Trk expressed in cells that are deficient in endogenous GM1 due to the mutation of GM1 synthase gene (NG-CR72 cells) is responsive to its ligand nerve growth factor and how genetic restoration of GM1 synthase gene by a stable transfection of the gene affects the function of the Trk protein. The data clearly showed that (1) confocal lazor microscopic studies disclosed NG-CR72 cells are really deficient in GM1, (2) stable transfection of GM1 synthase cDNA into these cells (NG-CR72G cells) restores the expression of GM1 in the cells, and (3) Trk protein is expressed in NG-CR72 cells but its location seemed not to be on the plasma membrane, whereas we clearly observed that the Trk protein is expressed on the plasma membrane in NG-CR72G cells. (4) NGF did not elicit the autophosphorylation of the Trk protein in GM1 deficient NG-CR72 cells but did elicit the activation of the Trk protein in NG-CR72G cells with an activation of mitogen activated protein kinase. These studies strongly suggested that GM1 is necessary for the normal expression of the Trk protein function and for normal targeting of the Trk protein to the plasma membrane.     

Molecular Detection,Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to Valivorax paradoxus, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge.Many scientists have used the rRNA approach (29, 30) to detect microbial populations and to describe the structures of microbial communities in various environments without isolating the component microorganisms. These studies have shown that most 16S ribosomal DNA (rDNA) sequences directly amplified from environmental samples are different from the sequences of comparable laboratory strains. Workers have concluded from such observations that many bacteria that are predominant in the natural environment have not been isolated in the laboratory yet and that the microbial diversity in the natural environment is much greater than the diversity of the bacteria that have been isolated (2, 7, 13, 25, 35, 36, 39, 40).Currently, one important aspect of microbial ecology studies is functional dissection of microbial communities based on structural information obtained by the approach mentioned above. An analysis of a population shift accompanied by a change in the function of a community yields information useful for identifying functionally dominant populations (2, 3, 42), although information concerning the function (activity) of each population can never be obtained by this kind of approach. Hence, workers have emphasized that pure-culture experiments are indispensable for detailed analysis of the functions of each population and that isolation of the functionally dominant populations in a microbial community is quite important.Phenol and its derivatives are some of the major hazardous compounds in industrial wastewater (1, 31, 43), and for this reason biodegradation of phenol has attracted keen attention (34, 46). However, since most studies of phenol biodegradation have been carried out under laboratory conditions with arbitrarily selected phenol-degrading bacteria, phenol biodegradation in the environment is not well understood yet. In the present study, to better understand phenol degradation in activated sludge, we isolated and characterized the phenol-degrading bacteria that were identified by the rRNA approach to be the dominant population in phenol-digesting activated sludge. Physiological and genetic differences between the dominant phenol-degrading bacteria isolated in this study and representative phenol-degrading bacteria characterized previously in several laboratories are discussed below.     

Cloning and Nucleotide Sequence of the gyrB Gene of Vibrio parahaemolyticus and Its Application in Detection of This Pathogen in Shrimp

Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of Vibrio parahaemolyticus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic probe. The gyrB genes of V. parahaemolyticus and closely related Vibrio alginolyticus were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within gyrB specific for V. parahaemolyticus. These primers recognized 117 of 117 reference and wild-type V. parahaemolyticus strains, whereas amplification did not occur when 90 strains of 37 other Vibrio species or 60 strains representing 34 different nonvibrio species were tested. In 100-μl PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of gyrB primers for the routine identification of V. parahaemolyticus in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp V. parahaemolyticus-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of V. parahaemolyticus cells per g of shrimp homogenate. By this approach, we were able to detect V. parahaemolyticus in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of V. parahaemolyticus in shrimp.     

Reduction of rat myocardial ischemia and reperfusion injury by sialyl Lewis x oligosaccharide and anti-rat P-selectin antibodies

Polymorphonuclear leukocytes (PMN) are directly involved indevelopment of ischemic myocardial injury. Adhesion of PMN toendothelial cells is an initial step that triggers a sequentialprocess leading to acute inflammatory responses. Interactionbetween P-selectin and its oligosaccharide ligand, sialyl Lewisx (sLe^x), plays an important role in the early stage of theadhesion. To examine the role of P-selectin in various animaldisease models especially in rats, we have cloned rat E- andP-selectin cDNAs and established monoclonal antibodies againstthese rat selectins. In this report, we describe the generationand characterization of anti-rat P-selectin antibodies (ARPs).These antibodies detect cell surface P-selectin on thrombin-stimulatedrat platelets. More importantly, intravenous administrationof ARP2-4 reduced infarction developed after 30 min of ischemiafollowed by 24 h of reperfusion in a rat myocardial injury model.In addition, similar protective effect was also observed byadministration of a sLe^x- oligosaccharide. These results indicatethat cell adhesion mediated via P-selectin is involved in thedevelopment of ischemia and reperfusion injury in rat heart. ischemia and reperfusion injury monoclonal antibodies selectins sialyl Lewis x     

Close phylogenetic relationship between vestimentifera (tube worms) and annelida revealed by the amino acid sequence of elongation factor-lα

To clarify the phylogenetic position of Vestimentifera (tube worms), 346-bp fragments of the elongation factor-l (EF-l) gene (939–1286 according to the numbering of the human gene) of a vestimentiferan, Lamellibrachia sp., a sternaspid polychaete, Sternaspis scutata, an earthworm, Pheretima sp., and a gastropod, Alviniconcha hessleri, were sequenced. From the amino acid sequences of these EF-l, and those of two other vertebrates and two arthropods, phylogenetic relationships were deduced by the maximum likelihood (ML) method, by which the phylogenetic tree can be inferred without assuming constancy of the molecular evolutionary rate. For the ML tree and all of seven alternative trees, whose log-likelihoods could not be discriminated from that of the ML tree by the criterion of the standard error, the vestimentiferan, the polychaete, and the oligochaete formed a clade, excluding the arthropods and the gastropod as outgroups. This result is convincing evidence that Vestimentifera are protostomes that are closely related to Annelida. The ML tree suggests that Vestimentifera are more closely related to Polychaeta than to Oligochaeta, though the data were not sufficient to discriminate these three groups at a significant level. From recent evidence such as morphological characteristics and molecular information, it may safely be said that vestimentiferans should be included in the Annelida provided this phylum contains polychaetes and oligochaetes.Correspondence to: S. Kojima     

Cas9-guide RNA ribonucleoprotein-induced genome editing in the industrial green alga <Emphasis Type="Italic">Coccomyxa</Emphasis> sp. strain KJ

Background

Oxygen-evolving photosynthetic microorganisms, collectively termed as microalgae, are gaining attention as alternative fuel sources. The unicellular alga Coccomyxa sp. strain KJ that belongs to the class Trebouxiophyceae can grow rapidly in minimal mineral media and accumulate triacylglycerols at levels?>?60% (w/w) of its dry weight under nitrogen depletion conditions. Thus, the strain can be a good candidate for biofuel production. Still, substantial improvements in lipid productivity and other traits of this strain are needed to meet commercial production requirements. Consequently, the development of new genetic tools including genome editing that are applicable to this strain is highly desired.

Results

In this paper, we report successful genome editing of strain KJ by intracellular delivery of a ribonucleoprotein complex comprising recombinant Cas9 protein and guide RNA. For introduction of Cas9-guide RNA ribonucleoprotein into strain KJ cells, we used an electroporator with a short (2.5 ms) electric pulse at a high field strength (7500 V cm^?1) followed by multiple 50-ms electric pulses at low field strength (250 V cm^?1). Under these conditions, we successfully isolated several knockout lines of the FTSY gene of strain KJ, encoding a signal recognition particle-docking protein at a frequency of 0.01%.

Conclusions

Our study shows applicability of DNA-free genome editing in Coccomyxa, which may be applicable in other Trebouxiophyceae species.

     

Ferritin forms dynamic oligomers to associate with microtubules in vivo: implication for the role of microtubules in iron metabolism

Ferritin, a ubiquitously distributed iron storage protein, has been reported to interact with microtubules in vitro (Hasan et al., 2005, FEBS journal 272:822-831). Here, we demonstrate that ferritin binds with the microtubules in an oligomeric form and that the microtubule-bound ferritin contains more than two-fold amount of iron compared to the unbound ferritin fraction in vitro. Indirect immunofluorescence microscopy showed that a significant fraction of the ferritin molecules colocalized with the microtubules as oligomers in a wide variety of cell lines. These findings are consistent with the immediate oligomerization of rhodamine-labeled ferritin, microinjected in living human hepatoma cells. Ferritin oligomers were dynamic in the cytoplasm, and an anti-microtubule drug significantly inhibited their intracellular movement. Treatment of cells with an iron donor, ferric ammonium citrate, remarkably increased the number of cells containing ferritin oligomers. On the other hand, when the cells, such as mouse neuroblastoma cells, were deprived of iron, ferritin oligomers were localized in the microtubule dense, neurite shafts, but were disappeared from the microtubule deficient neurite tips. These data indicate that the microtubules provide a scaffold for the cytoplasmic distribution and transport of the iron-rich ferritin and implicate the role of microtubules in iron metabolism.