Studies on the ɛ-Lysine Acylase

?-Lysine acylase of Achromobacter pestifer EA was purified by fractionations with ammonium sulfate and acetone, and by vertical zone electrophoresis. As a result, this bacterial ?-lysine acylase was obtained as an electrophoretically homogeneous protein, specific activity of which is the highest among ?-lysine acylases ever reported.     

Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments

Summary Various DNA subfragments were derived from miniF DNA by complete or partial PstI cleavage, and cloned in the plasmid vectors pBR322 or dv1. The recombinant plasmids obtained were introduced into an Escherichia coli minicell-producing strain, and the plasmid-coded proteins were radiolabeled and analyzed by gel electrophoresis. Six miniF-encoded proteins, larger than 11 000 daltons, were detected and their coding regions were mapped on the F plasmid genome. Three of them were assigned by taking into account the known nucleotide sequences (Murotsu et al. 1981; K. Yoshioka, personal communication). The coding directions of some proteins were determined by inserting the lac promotor into one of the recombinant plasmids and analyzing the increase in production of the proteins. The coding direction of the five proteins analyzed so far was uniform. Comparison of these results with a functional map of miniF suggested possible roles of the proteins.     

Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection

A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappaB (NF-kappaB), has been developed by using a DNA-detectable chemiluminogenic reagent and a centrifugal filter that distinguishes different molecular sizes. After the formation of a complex between NF-kappaB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappaB is estimated by chemiluminescence detection of the bound DNA. This detection is performed within 2 min at room temperature by the use of a chemiluminogenic reagent, 3',4',5'-trimethoxyphenylglyoxal, which selectively recognizes guanine moiety in oligonucleotides or DNAs. This method does not require any labeled probes or antibodies and can determine a concentration as low as 5 nM of DNA-binding NF-kappaB. The sensitivity is nearly the same as that of other methods such as gel shift assay using fluorescence-labeled probes and enzyme-linked immunosorbent assay. Therefore, the current method provides a convenient tool for surveying various DNA-binding proteins.     

Exercise causes tissue-specific enhancement of endothelin-1 mRNA expression in internal organs

Endothelin-1 (ET-1) is a potent vasoconstrictorpeptide, which also potentiates contractions to norepinephrine in humaninternal mammary and coronary vessels. Exercise causes a redistribution of blood flow, i.e., the increase in working muscles that is partly attributable to a decrease in visceral blood flow. We hypothesized thatexercise causes a tissue-specific increase in ET-1 expression ininternal organs. We studied whether exercise affects expression ofpreproET-1 mRNA in the kidneys and lungs. The rats performed treadmillrunning (0% grade) for 45 min at a speed of 25 m/min. The plasmaconcentrations of ET-1, epinephrine, and norepinephrine were greater inthe exercise rats than in the sedentary control rats. The expression ofpreproET-1 mRNA in the kidneys was markedly higher in the exercise ratsthan in the sedentary control rats, whereas that in the lungs did notdiffer between the two groups. Therefore, the present study provides apossibility that the exercise-induced increase in production of ET-1 inthe kidneys causes vasoconstriction and hence decreases blood flow inthe kidneys through its direct vasoconstrictive action and/orits indirect effect of enhancing vasoconstrictions to norepinephrine.

     

Feedback controls restrain the initiation of Escherichia coli chromosomal replication

In Escherichia coli, initiation of chromosomal replication is activated by a nucleoprotein complex formed primarily between the DnaA protein and oriC (replication origin) DNA. After replicational initiation, this complex has to be inactivated in order to repress the appearance of initiation events until the next scheduled round of initiation. Studies of the mechanisms responsible for this repression have recently revealed direct coupling between these mechanisms and key elements of the replication process, suggesting that feedback-type regulatory loops exist between the factors implicated in initiation and the elements yielded by the replication process. The loading of the ring-shaped beta-subunit of DNA polymerase III onto DNA plays a key role in the inactivation of the DnaA protein. Duplication of oriC DNA results in hemimethylated DNA, which is inert for reinitiation. Titration of large amounts of DnaA protein to a non-oriC locus can repress untimely initiations, and timely duplication of this locus is required for this repression in rapidly growing cells. All these systems functionally complement one another to ensure the maintenance of the interinitiation interval between two normal DNA replication cycles. The mechanisms that link the replication cycle to the progression of the cell cycle are also discussed.     

Combined brain‐derived neurotrophic factor with extinction training alleviate impaired fear extinction in an animal model of post‐traumatic stress disorder

Impaired fear memory extinction (Ext) is one of the hallmark symptoms of post‐traumatic stress disorder (PTSD). However, since the precise mechanism of impaired Ext remains unknown, effective interventions have not yet been established. Recently, hippocampal‐prefrontal brain‐derived neurotrophic factor (BDNF) activity was shown to be crucial for Ext in naïve rats. We therefore examined whether decreased hippocampal‐prefrontal BDNF activity is also involved in the Ext of rats subjected to a single prolonged stress (SPS) as a model of PTSD. BDNF levels were measured by enzyme‐linked immunosorbent assay (ELISA), and phosphorylation of TrkB was measured by immunohistochemistry in the hippocampus and medial prefrontal cortex (mPFC) of SPS rats. We also examined whether BDNF infusion into the ventral mPFC or hippocampus alleviated the impaired Ext of SPS rats in the contextual fear conditioning paradigm. SPS significantly decreased the levels of BDNF in both the hippocampus and mPFC and TrkB phosphorylation in the ventral mPFC. Infusion of BDNF 24 hours after conditioning in the infralimbic cortex (ILC), but not the prelimbic cortex (PLC) nor hippocampus, alleviated the impairment of Ext. Since amelioration of impaired Ext by BDNF infusion did not occur without extinction training, it seems the two interventions must occur consecutively to alleviate impaired Ext. Additionally, BDNF infusion markedly increased TrkB phosphorylation in the ILC of SPS rats. These findings suggest that decreased BDNF signal transduction might be involved in the impaired Ext of SPS rats, and that activation of the BDNF‐TrkB signal might be a novel therapeutic strategy for the impaired Ext by stress.     

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.     

Proteome analysis of programmed cell death and defense signaling using the rice lesion mimic mutant cdr2

We have previously identified three lesion-mimic mutants, cell death and resistance (cdr), in rice. These mutants induce a series of defense responses, including expression of defense-related genes and high accumulation of phytoalexins, indicating that the cdr mutants are useful materials to study programmed cell death and defense signaling in rice. Here, we carried out a proteome analysis of the cdr2 mutant. Total proteins prepared from the wild type and the cdr2 mutant at three different stages of lesion formation were compared using two-dimensional electrophoresis. We found a total of 37 proteins that were differentially expressed between cdr2 and wild type. Among them, 28 spots were up-regulated and nine were down-regulated in the cdr2 mutant. All the protein spots were identified by mass spectrometric analysis. These differentially regulated proteins included defense-related proteins. In addition, 27 proteins were classified as metabolic enzymes, suggesting that the programmed cell death that occurs in the cdr2 mutant is associated with active metabolic changes. Our study shows that proteome analysis is a useful approach to study programmed cell death and defense signaling in plants.     

Versican Facilitates Chondrocyte Differentiation and Regulates Joint Morphogenesis

Versican/PG-M is a large chondroitin sulfate proteoglycan in the extracellular matrix, which is transiently expressed in mesenchymal condensation areas during tissue morphogenesis. Here, we generated versican conditional knock-out mice Prx1-Cre/Vcan^flox/flox, in which Vcan is pruned out by site-specific Cre recombinase driven by the Prx1 promoter. Although Prx1-Cre/Vcan^flox/flox mice are viable and fertile, they develop distorted digits. Histological analysis of newborn mice reveals hypertrophic chondrocytic nodules in cartilage, tilting of the joint, and a slight delay of chondrocyte differentiation in digits. By immunostaining, whereas the joint interzone of Prx1-Cre/Vcan^+/+ shows an accumulation of TGF-β, concomitant with versican, that of Prx1-Cre/Vcan^flox/flox without versican expression exhibits a decreased incorporation of TGF-β. In a micromass culture system of mesenchymal cells from limb bud, whereas TGF-β and versican are co-localized in the perinodular regions of developing cartilage in Prx1-Cre/Vcan^+/+, TGF-β is widely distributed in Prx1-Cre/Vcan^flox/flox. These results suggest that versican facilitates chondrogenesis and joint morphogenesis, by localizing TGF-β in the extracellular matrix and regulating its signaling.