Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia

Venom of an Australian ant species of the Myrmecia pilosula species complex (mss. name Myrmecia banksi Taylor) contains two major allergenic peptides, pilosulin 1 and pilosulin 2. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of another Myrmecia species was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulin 1 and pilosulin 2. As a result, we isolated cDNA clones encoding the novel antimicrobial peptides pilosulin 3 and pilosulin 4. The nucleotide and the amino acid sequences of all four pilosulins have high homology except for the mature peptide coding regions. Synthetic pilosulin 3 and pilosulin 4 peptides displayed antimicrobial activity with histamine-releasing and low hemolytic activities.     

The FHA domain of aprataxin interacts with the C-terminal region of XRCC1

Aprataxin (APTX) is the causative gene product for early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH/AOA1). In our previous study, we found that APTX interacts with X-ray repair cross-complementing group 1 (XRCC1), a scaffold protein with an essential role in single-strand DNA break repair (SSBR). To further characterize the functions of APTX, we determined the domains of APTX and XRCC1 required for the interaction. We demonstrated that the 20 N-terminal amino acids of the FHA domain of APTX are important for its interaction with the C-terminal region (residues 492-574) of XRCC1. Moreover, we found that poly (ADP-ribose) polymerase-1 (PARP-1) is also co-immunoprecipitated with APTX. These findings suggest that APTX, together with XRCC1 and PARP-1, plays an essential role in SSBR.     

Identification of a G protein-coupled receptor specifically responsive to beta-alanine

We isolated a cDNA encoding an orphan G protein-coupled receptor, TGR7, which has been recently reported to correspond to MrgD. To search for ligands for TGR7, we screened a series of small molecule compounds by detecting the Ca2+ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that beta-alanine at micromolar doses specifically evoked Ca2+ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, gamma-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, beta-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins Gq and Gi. In guanosine 5'-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, beta-alanine specifically increased the binding of guanosine 5'-O-3-thiotriphosphate. When a fusion protein composed of TGR7 and green fluorescent protein was expressed in cells, it localized at the plasma membrane but internalized into the cytoplasm after treatment with beta-alanine. In addition, we found that beta-[3H]alanine more efficiently bound to TGR7-expressing cells than to control cells. From these results, we concluded that TGR7 functioned as a specific membrane receptor for beta-alanine. Quantitative PCR analysis revealed that TGR7 mRNA was predominantly expressed in the dorsal root ganglia in rats. By in situ hybridization and immunostaining, we confirmed that TGR7 mRNA was co-expressed in the small diameter neurons with P2X3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain.     

Teratogenic effects of bis-diamine on the developing cardiac conduction system

BACKGROUND: Congenital heart defects, including conotruncal anomalies, are often associated with arrhythmias. Bis-diamine induces conotruncal anomalies in embryos when administered to pregnant female rats. To investigate the mechanism of arrhythmia in conotruncal anomalies, we histologically examined the development of the cardiac conduction system in this animal model. METHODS: A single dose of 200 mg of bis-diamine was administered to pregnant Wistar rats on ED 10.5 of pregnancy. The embryos were removed on each day from ED 11.5 to 15.5. Immunoexpression of HNK-1, connexin40, and connexin43 were examined in serial sections. The distribution pattern of TUNEL-positive cells around the conduction system was also examined. RESULTS: HNK-1 immunoreactivity was evident in interventricular septum, in both the control and the bis-diamine-treated embryos from ED 12.5. Although a chain of connexin40-immunoreactive cells from interventricular septum to trabeculae, corresponding to the His bundle and its branches, was demonstrated at ED 13.5 in the control embryos, this chain was first detected at ED 14.5 in the bis-diamine-treated embryos. Immunoexpression of connexin43 in the working myocardium was also less in the bis-diamine-treated embryos than in the control at ED 13.5. The number of TUNEL-positive cells in the interventricular septum was highest at ED 12.5 in the control and at ED 13.5 in the bis-diamine-treated embryos. Furthermore, these TUNEL-positive cells were HNK-1 negative, vimentin-positive, and alpha smooth muscle actin-positive. CONCLUSIONS: Bis-diamine disturbed the normal development of gap junctions and apoptosis of myofibroblasts around the HNK-1-positive conduction tissue through overall poor myocardial proliferation and growth.     

Age-related changes in the expression of gelatinase and tissue inhibitor of metalloproteinase genes in mandibular condylar,growth plate,and articular cartilage in rats

Summary Mandibular condylar cartilage acts as both articular and growth plate cartilage during growth, and then becomes articular cartilage after growth is complete. Cartilaginous extracellular matrix is remodeled continuously via a combination of production, degradation by matrix metalloproteinases (MMPs), and inhibition of MMP activity by tissue inhibitors of metalloproteinases (TIMPs). This study attempted to clarify the age-related changes in the mRNA expression patterns of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in mandibular condylar cartilage in comparison to tibial growth plate and articular cartilage using an in situ hybridization method in growing and adult rats. MMP-2 and MMP-9 were expressed in a wide range of condylar cartilage cells during growth, and their expression domains became limited to mature chondrocytes in adults. The patterns of TIMP-1 and TIMP-2 expression were similar to those of MMP-2 and MMP-9 during growth, and were maintained until adulthood. TIMP-3 was localized to hypertrophic chondrocytes throughout the growth stage. Therefore, we concluded that TIMP-1 and TIMP-2 were general inhibitors of MMP-2 and MMP-9 in condylar cartilage, while TIMP-3 regulates the collagenolytic degradation of the hypertrophic cartilage matrix.     

Intermolecular and intramolecular readout mechanisms in protein-DNA recognition

Protein-DNA recognition plays an essential role in the regulation of gene expression. Regulatory proteins are known to recognize specific DNA sequences directly through atomic contacts (intermolecular readout) and/or indirectly through the conformational properties of the DNA (intramolecular readout). However, little is known about the respective contributions made by these so-called direct and indirect readout mechanisms. We addressed this question by making use of information extracted from a structural database containing many protein-DNA complexes. We quantified the specificity of intermolecular (direct) readout by statistical analysis of base-amino acid interactions within protein-DNA complexes. The specificity of the intramolecular (indirect) readout due to DNA was quantified by statistical analysis of the sequence-dependent DNA conformation. Systematic comparison of these specificities in a large number of protein-DNA complexes revealed that both intermolecular and intramolecular readouts contribute to the specificity of protein-DNA recognition, and that their relative contributions vary depending upon the protein-DNA complexes. We demonstrated that combination of the intermolecular and intramolecular energies derived from the statistical analyses lead to enhanced specificity, and that the combined energy could explain experimental data on binding affinity changes caused by base mutations. These results provided new insight into the relationship between specificity and structure in the process of protein-DNA recognition, which would lead to prediction of specific protein-DNA binding sites.     

Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1

We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana. The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs. Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation. The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast. Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker. These findings indicate for the first time that OsTPC1 is a putative VDCC in rice.     

Nucleotide sequencing analysis of the swine 433-kb genomic segment located between the non-classical and classical<Emphasis Type="Italic"> SLA</Emphasis> class I gene clusters

Genome analysis of the swine leukocyte antigen (SLA) region is needed to obtain information on the MHC genomic sequence similarities and differences between the swine and human, given the possible use of swine organs for xenotransplantation. Here, the genomic sequences of a 433-kb segment located between the non-classical and classical SLA class I gene clusters were determined and analyzed for gene organization and contents of repetitive sequences. The genomic organization and diversity of this swine non-class I gene region was compared with the orthologous region of the human leukocyte antigen (HLA) complex. The length of the fully sequenced SLA genomic segment was 433 kb compared with 595 kb in the corresponding HLA class I region. This 162-kb difference in size between the swine and human genomic segments can be explained by indel activity, and the greater variety and density of repetitive sequences within the human MHC. Twenty-one swine genes with strong sequence similarity to the corresponding human genes were identified, with the gene order from the centromere to telomere of HCR - SPR1 - SEEK1 - CDSN - STG - DPCR1 - KIAA1885 - TFIIH - DDR - IER3 - FLOT1 - TUBB - KIAA0170 - NRM - KIAA1949 - DDX16 - FLJ13158 - MRPS18B - FB19 - ABCFI - CAT56. The human SEEK1 and DPCR1 genes are pseudogenes in swine. We conclude that the swine non-class I gene region that we have sequenced is highly conserved and therefore homologous to the corresponding region located between the HLA-C and HLA-E genes in the human.The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL and GenBank databases under accession numbers AB113354, AB113355, AB113356, AB113357     

coq7/clk-1 regulates mitochondrial respiration and the generation of reactive oxygen species via coenzyme Q

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, and shows sluggish behaviours and an extended lifespan. In C. elegans and Saccharomyces cerevisiae, coq7/clk-1 is required for the biosynthesis of coenzyme Q (CoQ), an essential co-factor in mitochondrial respiration. The clk-1 mutant contains dietary CoQ(8) from Escherichia coli and demethoxyubiquinone 9 (DMQ9) instead of CoQ(9). In a previous study, we generated COQ7-deficient mice by targeted disruption of the coq7 gene and reported that mouse coq7/clk-1 is also essential for CoQ synthesis, maintenance of mitochondrial integrity and neurogenesis. In the present study, we rescued COQ7-deficient mice from embryonic lethality and established a mouse model with decreased CoQ level by transgene expression of COQ7/CLK-1. A biochemical analysis showed a concomitant decrease in CoQ(9), mitochondrial respiratory enzyme activity and the generation of reactive oxygen species (ROS) in the mitochondria of CoQ-insufficient mice. This implied that the depressed activity of respiratory enzymes and the depressed production of ROS may play a physiological role in the control of lifespan in mammalian species and of C. elegans.     

Analysis of ascidian <Emphasis Type="Italic">Not</Emphasis> genes highlights their evolutionarily conserved and derived features of structure and expression in development

The ascidian larva is often regarded as an organism close to the ancestral form of chordates, while it is generally accepted that the Spemanns organizer is absent from ascidian embryos. Not is one of the genes expressed in the organizer to execute functions in vertebrate embryos. To address the extent of conservation of Not gene expression among ascidians and vertebrates, we examined the structure and developmental expression of Not of the two distantly related ascidian species, Halocynthia and Ciona. Putative ascidian Not proteins were noted by the absence of one of the two motifs conserved among Not proteins of sea urchin and vertebrates. Analysis by in situ hybridization revealed that Not gene expression of ascidians could be categorized into three types: expression likely to be conserved between ascidians and vertebrates, that probably unique to ascidians, and that specific to ascidian species. Expression of ascidian Not in the posterior end of the tail as well as the notochord and a small part of the anterior neural tube at the tailbud stage is reminiscent of the expression of the vertebrate counterparts in the tailbud, which is regarded as a continuation of the organizer and the pineal gland, respectively. The expression of Not in the epidermis precursors during cleavage stage may be unique to ascidians. In the light of the present findings, evolutionary aspects of Not genes are discussed.Electronic Supplementary Material Supplementary material is available for this article at Edited by N. Satoh