Endogenous protein and enzyme fragments induce immunoglobulin E-independent activation of mast cells via a G protein-coupled receptor, MRGPRX2.

Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through 2 main pathways, immunoglobulin E-dependent and E-independent activation. In the latter pathway, mast cells are activated by a diverse range of basic molecules (collectively known as basic secretagogues) through Mas-related G protein-coupled receptors (MRGPRs). In addition to the known basic secretagogues, here, we discovered several endogenous protein and enzyme fragments (such as chaperonin-10 fragment) that act as bioactive peptides and induce immunoglobulin E-independent mast cell activation via MRGPRX2 (previously known as MrgX2), leading to the degranulation of mast cells. We discuss the possibility that MRGPRX2 responds various as-yet-unidentified endogenous ligands that have specific characteristics, and propose that MRGPRX2 plays an important role in regulating inflammatory responses to endogenous harmful stimuli, such as protein breakdown products released from damaged or dying cells.

Repeated haemorrhages in peripheral nerve sheath tumours of the salivary glands after minor injury.

Repeated haemorrhages in peripheral nerve sheath tumours of the salivary glands are rare. We report the case of a patient with neurofibromatosis type 1 who had two episodes of massive haemorrhage in his right parotid gland the day after a minor injury. Oral and maxillofacial surgeons should be aware that vasculopathy may occur in patients with these tumours.

The transcriptional landscape of the mammalian genome.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Profiling of proteins associated with cisplatin resistance in ovarian cancer cells.

Resistance to cisplatin is a major impediment to the successful treatment of ovarian cancer, but the precise nature of the resistance is still unclear. In the current study, we aimed to investigate and compare the protein expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. We employed the recent development of surface-enhanced laser desorption/ionization ProteinChip technology to measure protein expression in three human ovarian cancer cell lines (KF-1, MN-1, and A2780) and their sublines (KF-r, MN-r, and A2780cp) resistant to cisplatin. The ProteinChip Arrays were analyzed using the ProteinChip Reader. We did not find any regularity in protein expressions in secretions of cisplatin-sensitive and cisplatin-resistant cells. But on the IMAC3 array, we captured 12 identical expressions which represent a subset of proteins whose expression levels are different between parent ovarian cancer cells and their cisplatin-resistant cells. In particular, at the molecular weight of 7829 d, three kinds of parent cell lines exhibited an elevated expression and their cisplatin-resistant sublines revealed a lowered expression. At the molecular weight of 6881 d, for KF and MN cell lines, opposite protein expressions were seen in the parent cell line and its cisplatin-resistant subline. We think the interesting protein expressions perhaps suggest some mechanisms involved in cisplatin resistance.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Functional annotation of a full-length mouse cDNA collection.

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

On-line coupling of solid-phase extraction to gas chromatography with fast solvent vaporization and concentration in an open injector liner. Analysis of pesticides in aqueous samples.

The purpose of this study is to combine solid-phase extraction (SPE) with gas chromatography (GC) for the fully automated determination of pesticides and herbicides in aqueous samples. The interface technique employed for connecting SPE with GC was fast solvent vaporization and concentration in an open injector liner. The interface device consisted of the programmed-temperature vaporizing injector without using the packing material in the liner and the target compounds were concentrated around the inlet of the GC capillary column. This avoided the degradation of target compounds, and no precise control of the injecting speed was required, when an automatic SPE system was connected to GC-MS. The aqueous samples used in this system were prepared by spiking 29 kinds of pesticide and herbicide compounds, which are regulated by the Ministry of Health and Welfare of the Japanese National government, in purified water and river water, to a resulting concentration of 1 microg/l. Employing this system, the recoveries and RSDs (n=6) of most compounds were greater than 75% and within 10%, respectively. From the results of this study, we found that on-line automatic SPE and capillary GC-MS equipped with the fast solvent vaporizing and concentrating method in an open injector liner could be connected in order to obtain good results for the determination of pesticides in water samples.

A cellular variant of supratentorial hemangioblastoma.

Supratentorial hemangioblastomas are rarely seen, especially in children and adolescents. We report the case of a 17-year-old male with supratentorial hemangioblastoma. Neuroimaging demonstrated a cystic lesion within the right parietal lobe. Systemic examination revealed no abnormality. The lesion was not attached to the dura and was not associated with von Hippel-Lindau disease. It was very difficult to confirm the final diagnosis of this case, in spite of extensive examination by light microscopy, immunohistochemical studies, and electron microscopy.

Characterization of a flagellar sheath component, PF60, and its structural gene in marine Vibrio.

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.

Suppression by the DNA fragment of the motX promoter region on long flagellar mutants of Vibrio alginolyticus.

The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5 microm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild-type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long-Pof strain V10578. The swimming speed and flagellar length of these transformants were almost equal to the wild-type values. The amounts of PF47 flagellin and PF60 sheath-associated protein, which increased in the long-Pof mutants, were retrieved to almost the wild-type level in the transformants. The plasmid pMF209 contained only a 143 bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long-Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.

Flagellin-containing membrane vesicles excreted from Vibrio alginolyticus mutants lacking a polar-flagellar filament.

Polar flagellum-defective mutants (Pof- Laf-) have been isolated from a lateral flagella-defective mutant (Pof+ Laf-). Among these Pof- Laf- mutants, polar-filamentless mutants, which have the hook structure but not the filament, were identified by electron microscopy. Their hooks were covered with a sheath structure which is contiguous to the outer membrane. The filament proteins, flagellins, were shed into the culture medium of these mutants. These flagellins could be sedimented by high-speed centrifugation even after heat or low pH treatment whereas the depolymerized flagellin of the Pof+ strain was degraded by these treatments. After Triton X-100 treatment, most flagellin of the filamentless mutants could no longer be sedimented, and was degraded. We observed vesicle-like structures on the tips of the hooks and in the flagellin fraction sedimented by high speed centrifugation. These results suggest that flagellin of the filamentless mutants is not assembled into the tip of the hook, but is excreted together with a membrane structure which is probably the sheath of polar flagella.

Structure and expression of bombyxin E1 gene: a novel family gene that encodes bombyxin-IV, an insect insulin-related neurosecretory peptide.

A bombyxin gene encoding precursor molecule for bombyxin-IV, one of the insulin-related neurosecretory peptide of the silkmoth Bombyx mori, has been cloned and characterized. The nucleotide sequence of this gene and its deduced amino acid sequence deviate moderately from those characterized previously for the family A, B, C and D bombyxin genes. The gene encoding the bombyxin-IV precursor was therefore defined into a novel family E and designated as gene E1. The bombyxin E1 transcript in Bombyx brain was shown to locate in four pairs of medial neurosecretory cells, which also produce other bombyxin family mRNAs, and the amount of the E1 transcript did not change markedly during the fifth larval instar. Genomic Southern hybridization indicated that the Bombyx haploid genome contained a single copy of the bombyxin family E gene.

Repressive regulation of the aquaporin-2 gene.

Expression of aquaporin-2 (AQP2) is exclusively limited to kidney collecting duct cells, and this strictly limited expression could be mediated by transcription of the gene. We first examined AQP2 mRNA expression in many cultured epithelial cells derived from kidney. Northern blot using OK, LLC-PK1, Madin-Darby canine kidney, and outer medullary collecting duct (OMCD) cells and primary culture of inner medullary collecting duct (IMCD) cells did not reveal any significant signal. A more sensitive method, ribonuclease protection assay, could detect a faint signal in OMCD cells when they were bathed in a hypertonic medium. Reverse-transcribed polymerase chain reaction applied to primary culture of IMCD cells showed a rapid dissipation of AQP2 mRNA within 4 days after culture. A reporter gene assay performed in the 1st day of primary culture of IMCD cells showed that the 5' region up to -2.9 kb worked as a promoter. Deletion experiments showed that at least two regions, from -434 to -364 and from -153 to -84, contain negatively acting cis-elements. When connected to a heterologous promoter, these regions repressed the activity in an orientation-dependent manner. These results suggest that transcription of AQP2 gene is strictly regulated and its ability is rapidly depressed in culture condition. This cell differentiation-specific expression of the gene may be, at least in part, mediated by the repressors present in its 5'-flanking region.

Traumatic basal ganglia haemorrhage: report of 7 cases.

We report 7 cases of traumatic basal ganglia haemorrhage (TBGH), which is closely related to diffuse axonal injury (DAI). Although most of our patients were in coma for more than 24 hours, the outcome was relatively favourable. All the patients regained consciousness, and 2 of 4 patients with contralateral hemiparesis showed improvement. Although expressive and nonfluent aphasia occurred in 5 patients, all of them recovered completely. The outcome in TBGH may be better than previously believed.

Improved crystals of the toxic protein MAP by protein engineering towards the host specificity.

Mirabilis anti-viral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F which exhibits a lower (1/100) activity against E. coli ribosomes while retaining almost full activity against mammalian cells [Habuka, Miyano, Kataoka, Tsuge & Noma (1992). J. Biol. Chem. 267, 7758-7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F MAP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the native MAP crystals [Miyano et al. (1992). J. Mol. Biol. 226, 281-283]: 50% ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3(1)21 (or P3(2)21) with a = b = 104.1 and c = 134.3 A. The crystals are isomorphous with the wild-type crystals but diffract to higher resolution. Imaging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.

Crystallization and preliminary X-ray crystallographic studies of recombinant human leukotriene A4 hydrolase complexed with bestatin.

Recombinant human leukotriene A4 hydrolase complexed with bestatin, an inhibitor of metalloprotease, has been crystallized by the hanging drop vapor diffusion method using 0.1 M phosphate buffer (pH 6.5) and 50 to 54% saturated ammonium sulfate. The orthorhombic crystals belong to the space group I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 273.6 A, b = 261.3 A and c = 52.9 A. They diffract beyond 2.5 A resolution and a native data set up to 3 A resolution has been collected on an imaging plate Weissenberg camera using synchrotron radiation.

Expression of a pokeweed antiviral protein in Escherichia coli and its characterization.

Two expression vectors were constructed to produce a putative mature alpha-pokeweed antiviral protein (alpha-PAP) in Escherichia coli with its NH2- and COOH-terminal extrapeptides excised. One was for its intracellular expression with a methionine at its NH2-terminal. The other was for its secretion using an ompA signal peptide. The former product was purified from the total soluble proteins of the transformant with a yield of 1.74 mg/liter and the latter had a yield of 5.55 mg/liter. Both products exhibited RNA N-glycosidase activity on wheat ribosomes and inhibitory activity to protein synthesis in a rabbit reticulocyte system.