A large deletion within intron 20 sequence of single-copy PolA1 gene as a useful marker for the speciation in Oryza AA-genome species.

Oryza AA-genome complex comprises five wild species, O. rufipogon, O. barthii, O. longistaminata, O. glumaepatula, and O. meridionalis. Evolutionary relationships among these five wild species have remained contentious and inconclusive. We found that intron 20 of PolA1, a single-copy nuclear gene, was short (S-type: 141-142 bp) in O. rufipogon, O. barthii, and O. glumaepatula, while long (L-type: ca. 1.5 kb) introns were apparent in O. longistaminata and O. meridionalis. Because Oryza species containing BB, CC, EE, FF, and GG genome showed L-type introns, the S-type intron was probably derived from the L-type intron by the deletion of a 1.4 kb fragment through intramolecular homologous recombination between two tandem TTTTGC repeats. Excluding the large deletion sequence, intron 20 sequence of O. barthii was identical to that of O. longistaminata. As more than 3,470 accessions of O. rufipogon and O. sativa also contained the same intron 20 sequence with O. longistaminata except for single T-nucleotide deletion, which was shared with O. glumaepatuala, the deletion of the T-nucleotide probably occurred in the L-type intron 20 of O. logistaminata. Deletions of a large 1.4 kb fragment and single T-nucleotide within the intron 20 of PolA1 gene were considered as useful DNA markers to study the evolutionary relationships among Oryza AA-genome species.

Genetic diversity of preserved rice seed samples from the Mikawa area, Japan, stored in the Meiji era.

Preserved rice (Oryza sativa L.) seeds stored for nearly a century as an emergency food stocks from the Mikawa area were investigated for their genetic diversity. Morphologically, the seeds appeared to be typical Japonica. One chloroplast INDEL petN-trnC, two nuclear INDELs Acp1 and Cat1, and three SNP markers in Starch synthase IIa were amplified to characterize the molecular profile. The efficiency of amplification varied among the markers. Most of preserved seeds were classified as Japonica, but some were identified as Indica. The heterozygous genotypes detected suggested a high frequency of outcrossing at that time. On the other hand, 21 SSR markers showed quite a high degree of amplification efficiency. Principal coordinate analysis and STRUCTURE analysis based on the SSR polymorphisms proved that the preserved seeds contained alleles that were not detected among current landraces and breeding varieties, and there were the expected three subpopulations among 96 preserved seeds. These results indicated that these preserved seeds from Mikawa area in Meiji era had high genetic diversity and consisted of some subpopulations including Indica landraces with typical Japonica seed shape. These lines were considered to have been lost from current genetic resources.

Genetic variation in rice (Oryza sativa L.) germplasm from northern Laos.

We assessed genetic variation in rice germplasm in northern Laos and Vientiane province from polymorphism data of SSR markers. We classified 314 accessions into three clusters; Ia (corresponding to the lowland Japonica Group), Ib (upland Japonica Group) and II (Indica Group). The accessions of cluster Ib grew mainly in mountainous fields, and those of cluster II grew commonly in basins and along rivers. The few accessions of cluster Ia grew in only three provinces: Houaphanh, Xiangkhouang and Vientiane. Lowland cultivars in cluster II were predominant in Vientiane. Variations in heading date under short-day conditions in 2014 and long-day conditions in 2015 indicate that many accessions were sensitive to the photoperiod on account of complex genetic mechanisms underlying both photoperiod sensitivity and basic vegetative growth. A total of 219 among whole accessions were classified into 6 groups: E1-3 and L1-3. E2 and E3 were dominant in clusters Ib and II; E1 and L1-3 were minor groups. These results demonstrate characteristic distributions of the Indica and Japonica Group's germplasms in northern Laos and their genetic variation in heading date.

Diverse genetic variation in maternal lineages with high heterogeneity among in situ-conserved wild rice (Oryza rufipogon Griff.) developed in Thailand.

Wild rice, Oryza rufipogon, is a genetic resource that can be used to improve cultivated rice, but its populations are now decreasing in terms of both size and number. Extensive research on wild rice has been conducted in Thailand, where two in situ conservation sites have been preserved in natural areas where perennial wild rice predominates. The genetic structure of wild rice populations was investigated by examining both the chloroplast and nucleus genomes at sites of in situ conservation site in Thailand. One accession from an in situ-conserved site was re-sequenced against the chloroplast genome of O. sativa cv. 'Nipponbare' to develop chloroplast insertion/deletion (cpINDEL) markers. These cpINDEL markers revealed unique maternal lineages in the in situ-conserved populations upon comparison with other Asian wild rice accessions. Diverse genetic variation was also detected with SSR markers throughout the genome. Three populations differed from each other and also within single populations. The sub-populations within an in situ-conserved population showed a complex population structure due to their multiple maternal lineages and relatively higher number of haplotypes when they maintained a relatively large population size. Such a heterogeneous population would serve as a unique gene pool for rice breeding.

Genetic diversity of the wx flanking region in rice landraces in northern Laos.

A glutinous texture of endosperm is one of the important traits of rice (Oyza sativa L.). Northern Laos is known as a center of glutinous rice diversity. We genotyped INDEL, SSR and SNP markers in a sample of 297 rice landraces collected in northern Laos. These glutinous varieties were confirmed to share a loss-of-function mutation in Granule bound starch synthase I (Wx). INDEL markers revealed a high frequency of recombinant genotypes between indica and japonica. Principal component analysis using SSR genotypes of Wx flanking region revealed that glutinous indica landraces were scattered between non-glutinous indica and glutinous-japonica types. High ratios of heterozygosity were found especially in glutinous indica. Haplotype analysis using SNP markers around Wx locus revealed that glutinous indica landraces would have a few chromosome segments of glutinous japonica. Frequent recombinations were confirmed outside of this region in glutinous indica. This intricate genetic structure of landraces suggested that glutinous indica landraces in Laos were generated through repeated natural crossing with glutinous-japonica landraces and severe selection by local farmers.

[Two cases of pancreatic cancer with multiple liver metastases treated with radical treatment after successful treatment with gemcitabine plus S-1 therapy].

UNLABELLED: We report two cases of pancreatic cancer with multiple liver metastases for which successful gemcitabine (GEM) +S-1 therapy facilitated radical resection. Case 1: A 40-year-old man with jaundice was diagnosed with pancreatic head cancer and multiple metastases. Radical treatment was not possible. Therefore, he was administered GEM +S-1 (GEM 1,000 mg/m² on days 1 and 8; S-1, 100mg/day for 14 days). Administration of seven courses of therapy achieved complete remission (CR) of the metastatic lesions. Considering the possibility of residual tumors, a subtotal stomach-preserving pancreaticoduodenectomy was performed. Although pathological examination revealed a residual tumor in the primary lesion, R0 resection was achieved. GEM+S-1 therapy was resumed, and the patient is currently relapse-free. Case 2: A 65-year-old man with obstructive jaundice was diagnosed with pancreatic head cancer and multiple metastases. Radical treatment was not possible. Therefore, GEM +S-1 therapy was initiated. After 11 therapy courses, only one metastatic lesion remained. Radical treatment was then deemed possible, and a pylorus-preserving pancreaticoduodenectomy was performed. Pathological examination revealed residual tumors in the primary lesion and the peripancreatic lymph nodes. However, the liver nodules were only fibrotic, and their surgical radicality was R0. GEM +S-1 therapy was resumed, and the patient is currently relapse-free. CONCLUSION: The high response rate of GEM+S-1 therapy suggests its usefulness in facilitating radical resection after treatment with (GEM) +S-1.

Role of desmoplakin in supporting neuronal activity, neurogenic processes, and emotional-related behaviors in the dentate gyrus.

Desmoplakin (Dsp) is a component of desmosomal cell-cell junctions that interacts with the cadherin complex and cytoskeletal intermediate filaments. In addition to its function as an adhesion component, Dsp is involved in various biological processes, such as gene expression, differentiation, and migration. Dsp is specifically expressed in the hippocampal dentate gyrus (DG) in the central nervous system. However, it is unclear how Dsp impacts hippocampal function and its related behaviors. Using an adeno-associated virus knockdown system in mice, we provide evidence that Dsp in the DG maintains hippocampal functions, including neuronal activity and adult neurogenesis, and contributes to anxiolytic-like effects. Dsp protein is mostly localized in mature granule cells in the adult DG. Dsp knockdown in the DG resulted in a lowered expression of an activity-dependent transcription factor FosB, and an increased expression of mature neuronal markers, such as calbindin. In addition, the suppression of Dsp decreases serotonin responsiveness at the DG output mossy fiber synapses and alters adult neurogenic processes in the subgranular zone of the DG. Moreover, DG-specific Dsp knockdown mice showed an increase in anxiety-like behaviors. Taken together, this research uncovers an unexplored function for Dsp in the central nervous system and suggests that Dsp in the DG may function as a regulator to maintain proper neuronal activation and adult neurogenesis, and contribute to the adaptation of emotion-related behavior.

Beckwith-Wiedemann syndrome with juvenile fibrous nodules and lobular breast tumors: a case report and review of the literature.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a genomic imprinting disorder caused by diverse genetic and/or epigenetic disorders of chromosome 11p15.5. BWS presents with a variety of clinical features, including overgrowth and an increased risk of embryonal tumors. Notably however, reports of patients with BWS and breast tumors are rare, and the association between these conditions is still unclear. Insulin-like growth factor-2 (IGF2) expression is known to be associated with the development of various cancers, including breast cancer, and patients with BWS with specific subtypes of molecular defects are known to show characteristic clinical features and IGF2 overexpression. CASE PRESENTATION: A 17-year-old girl who had been diagnosed with BWS based on an umbilical hernia, hyperinsulinemia, and left hemihypertrophy at birth, visited our department with a gradually swelling left breast. Her left breast was markedly larger than her right breast on visual examination. Imaging examinations showed two tumors measuring about 10 cm each in the left breast, and she was diagnosed with juvenile fibroadenoma following core needle biopsy. The two breast tumors were removed surgically and the patient remained alive with no recurrence. The final diagnosis was juvenile fibroadenoma without malignant findings. Immunohistochemical staining using IGF2 antibody revealed overexpression of IGF2 in the cytoplasm of ductal epithelial cells. Because of her clinical features and IGF2 overexpression, molecular defects of 11p15.5 including a possible genetic background of paternal uniparental disomy of chromosome 11 or hypermethylation of imprinting center 1 was suspected. CONCLUSIONS: In this case, overexpression of IGF2 suggested a possible relationship between BWS and breast tumors. Moreover, the characteristic clinical features and IGF2 staining predicted the subtype of 11p15.5 molecular defects in this patient.

Diversification and genetic differentiation of cultivated melon inferred from sequence polymorphism in the chloroplast genome.

Molecular analysis encouraged discovery of genetic diversity and relationships of cultivated melon (Cucumis melo L.). We sequenced nine inter- and intra-genic regions of the chloroplast genome, about 5500 bp, using 60 melon accessions and six reference accessions of wild species of Cucumis to show intra-specific variation of the chloroplast genome. Sequence polymorphisms were detected among melon accessions and other Cucumis species, indicating intra-specific diversification of the chloroplast genome. Melon accessions were classified into three subclusters by cytoplasm type and then into 12 subgroups. Geographical origin and seed size also differed between the three subclusters. Subcluster Ia contained small-seed melon from Southern Africa and South and East Asia and subcluster Ib mainly consisted of large-seed melon from northern Africa, Europe and USA. Melon accessions of subcluster Ic were only found in West, Central and Southern Africa. Our results indicated that European melon groups and Asian melon groups diversified independently and shared the same maternal lineage with northern African large-seed melon and Southern African small-seed melon, respectively. Cultivated melon of subcluster Ic may have been domesticated independently in Africa. The presence of 11 cytoplasm types in Africa strongly supported African origin of cultivated melon and indicated the importance of germplasm from Africa.

An adenocarcinoma in an inverted Meckel's diverticulum with intussusception.

BACKGROUND: Adenocarcinoma in an inverted Meckel's diverticulum with intussusception has not been reported to date. We discuss the clinical issues concerning this rare condition and review the relevant literature. CASE PRESENTATION: A 71-year-old Japanese female was referred to our hospital for further investigation of severe anemia. Computed tomography revealed a tumorous lesion in the terminal ileum. Capsule endoscopy did not provide detailed images. Exploratory laparoscopy revealed intussusception in the terminal ileum. An intraluminal tumor 70 cm proximal to the ileocecal valve was observed to be the lead point. Partial resection including the tumor was performed. Macroscopically, a polypoid tumor at the tip of an inverted diverticulum-like structure was observed. The tumor was histologically composed of adenocarcinoma accompanied by gastric and pyloric gland metaplasia in the background mucosa, which was confirmed by immunohistochemical staining. Based on these characteristics, this tumor is considered to have developed from the ectopic gastric mucosa in a Meckel's diverticulum. CONCLUSIONS: When we encounter patients with unfamiliar lesions in the small bowel, we need to differentiate Meckel's diverticulum related disease. Meckel's diverticulum can invert into the lumen of the small bowel and cause an intussusception, and has potential of malignant transformation.

Efficacy of Prophylactic Negative-Pressure Wound Therapy with Delayed Primary Closure for Contaminated Abdominal Wounds.

Background: Prophylactic negative-pressure wound therapy (NPWT) to prevent surgical site infection (SSI) may be effective for severely contaminated wounds. We investigated the safety and efficacy of NPWT with delayed primary closure (DPC) for preventing SSI. Methods: For patients with contaminated and dirty/infected surgical wounds after an emergency laparotomy, the abdominal fascia was closed with antibacterial absorbent threads and the skin was left open. Negative pressure (-80 mmHg) was applied through the polyurethane foam, which was replaced on postoperative days 3 and 7. DPC was performed when sufficient granulation was observed. The duration and adverse events of NPWT, the development of SSI, and the postoperative hospital stay were retrospectively reviewed. Results: We analyzed the cases of patients with contaminated (n = 15) and dirty/infected wounds (n = 7). The median duration of NPWT was 7 days (range 5-11 days). NPWT was discontinued in one (4.5%) patient due to wound traction pain. SSI developed in seven patients (31.8%), with incisional SSI in one (4.5%) and organ/space SSI in six (27.3%). The median postoperative hospital stay was 17 days (range 7-91 days). There was no significant relationship between postoperative hospital stay and wound classification (P=0.17) or type of SSI (P=0.07). Conclusion: Prophylactic NPWT with DPC was feasible and may be particularly suitable for severely contaminated wounds, with a low incidence of incisional SSI.

The rs8099917 polymorphism, when determined by a suitable genotyping method, is a better predictor for response to pegylated alpha interferon/ribavirin therapy in Japanese patients than other single nucleotide polymorphisms associated with interleukin-28B.

We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.

Raman Fingerprints of Rice Nutritional Quality: A Comparison between Japanese Koshihikari and Internationally Renowned Cultivars.

Raman spectroscopy was applied to characterize at the molecular scale the nutritional quality of the Japanese Koshihikari rice cultivar in comparison with other renowned rice cultivars including Carnaroli from Italy, Calrose from the USA, Jasmine rice from Thailand, and Basmati from both India and Pakistan. For comparison, two glutinous (mochigome) cultivars were also investigated. Calibrated and validated Raman analytical algorithms allowed quantitative determinations of: (i) amylopectin and amylose concentrations, (ii) fractions of aromatic amino acids, and (iii) protein content and secondary structure. The Raman assessments non-destructively linked the molecular composition of grains to key nutritional parameters and revealed a complex intertwine of chemical properties. The Koshihikari cultivar was rich in proteins (but with low statistical relevance as compared to other investigated cultivars) and aromatic amino acids. However, it also induced a clearly higher glycemic impact as compared to long-grain cultivars from Asian countries. Complementary to genomics and wet-chemistry analyses, Raman spectroscopy makes non-destructively available factual and data-driven information on rice nutritional characteristics, thus providing customers, dietitian nutritionists, and producers with a solid science-consolidated platform.

Feasibility and educational value of fluorescence cholangiography in laparoscopic cholecystectomy.

INTRODUCTION: Near-infrared fluorescence cholangiography during a laparoscopic cholecystectomy has become widely accepted as a useful auxiliary tool to visualize the extrahepatic biliary structures. We investigated the feasibility and educational value of a method with longer interval between the administration of indocyanine green and the imaging of these structures. METHODS: Approximately 18 hours before their surgery, patients (n = 51) were intravenously administered 0.25 mg/kg of indocyanine green. Each laparoscopic cholecystectomy was performed under fluorescence imaging in combination with white-light imaging. Operative outcomes including visualization of the extrahepatic biliary structures and operative time were compared between the patients on whom board-certified surgeons operated (feasibility phase; n = 18) and the patients on whom a surgery resident operated (educational phase; n = 33). RESULTS: There were no adverse events related to the longer interval method. The visualization rates of extrahepatic biliary structures were comparable between the two phases. Both the mean time to divide the cystic duct and the mean time to remove the gallbladder in the educational phase were significantly longer than those in the feasibility phase (68.2 vs 24.4 minutes and 30.2 vs 15.8 minutes, P < .001 each). There was no significant difference in other operative outcomes. The operative time learning curve did not decrease with a resident's experience. CONCLUSIONS: Fluorescence cholangiography with the longer interval method was feasible and could identify the extrahepatic biliary structures irrespective of the surgeon's experience; however, it did not decrease the operative time with experience.

Raman spectroscopic analysis of polysaccharides in popular Japanese rice cultivars.

Analytical algorithms based on Raman spectroscopy are proposed for the determination of amylopectin and amylose concentrations in polished white rice, and applied to characterize and compare linear and branched polysaccharide structures in nine different types of Japanese rice. A selected algorithm used symmetric bending vibrations of the COC glycosidic linkage from a relatively narrow spectral zone between 830 and 895 cm-1. It specifically compared the intensity of Raman signals from two types of bending common to both starch components (C1-O-C5 and C1-O-C4 at 868 and 855 cm-1, respectively) and that at the branch point peculiar to amylopectin (C1-O-C6 at 844 cm-1). Raman data were confronted with data collected by conventional amylose-iodine colorimetry method. Consistency was found between Raman and colorimetric methods over the entire series of tested rice cultivars, thus validating the newly proposed spectroscopic algorithm. The amylose content of the tested rice species broadly varied between 1.2 and 20.4%. The proposed Raman algorithm allows fast and nondestructive determination of amylose content in rice with minimal sample preparation. These characteristics might be key in the development of portable Raman devices capable to promptly screen polysaccharides in different rice cultivars with respect to their interannual and plantation-related fluctuations.

Primary malignant melanoma of the female urethra: A case report.

Primary malignant melanoma of a female urethra is extremely rare. A 71-year-old female was referred to our hospital with bleeding from the urethral meatus. Magnetic resonance imaging revealed a mass of 25 mm in diameter at the external urethral meatus. She underwent anterior pelvic exenteration and ileal conduit diversion. Histopathological findings were urethral malignant melanoma, pT3apN0M0 (stage IIA). Nine months after surgery, computed tomography revealed a local recurrence in the pelvis, the patient was treated with immunotherapy using combined nivolumab and ipilimumab; however, she did not respond to treatment and died 22 months following diagnosis.

Vitamin D Deficiency-induced Osteomalacia in a Patient with Anorexia Nervosa.

A 48-year-old woman with a 9-year-history of anorexia nervosa (AN) was admitted complaining of generalized bone pain. Blood tests showed hypocalcemia and hyperphosphatasemia, and a radiological survey revealed multiple rib fractures, suggesting complication with osteomalacia. Two years earlier, she had undergone subtotal colectomy for colon cancer. Her serum 25-hydroxy vitamin D concentration was below the detectable level. In addition to a poor nutritional intake and insufficient sun exposure, malabsorption of fat-soluble substances in the intestine and phosphate loss from the kidneys might have contributed to the development of her osteomalacia.

Raman Molecular Fingerprints of Rice Nutritional Quality and the Concept of Raman Barcode.

The nutritional quality of rice is contingent on a wide spectrum of biochemical characteristics, which essentially depend on rice genome, but are also greatly affected by growing/environmental conditions and aging during storage. The genetic basis and related identification of genes have widely been studied and rationally linked to accumulation of micronutrients in grains. However, genetic classifications cannot catch quality fluctuations arising from interannual, environmental, and storage conditions. Here, we propose a quantitative spectroscopic approach to analyze rice nutritional quality based on Raman spectroscopy, and disclose analytical algorithms for the determination of: (i) amylopectin and amylose concentrations, (ii) aromatic amino acids, (iii) protein content and structure, and (iv) chemical residues. The proposed Raman algorithms directly link to the molecular composition of grains and allow fast/non-destructive determination of key nutritional parameters with minimal sample preparation. Building upon spectroscopic information at the molecular level, we newly propose to represent the nutritional quality of labeled rice products with a barcode specially tailored on the Raman spectrum. The Raman barcode, which can be stored in databases promptly consultable with barcode scanners, could be linked to diet applications (apps) to enable a rapid, factual, and unequivocal product identification based on direct molecular screening.

Inhibition of prostaglandin D synthase suppresses muscular necrosis.

Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D(2). In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D(2) signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy. We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor beta1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D(2) production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy.

Expression, purification and characterization of leukotriene B(4) receptor, BLT1 in Pichia pastoris.

The high yield expression of BLT1, a G-protein coupled receptor for leukotriene B(4), was established in Pichia pastoris for structural studies. Guinea pig BLT1 was expressed in a functional form without post-translational modifications for the rapid purification and the crystallization. Among the BLT1s from four species, only guinea pig BLT1 was successfully expressed with the comparable binding affinity to BLT1 of native guinea pig tissues for several ligands. Only Asn4 of the two putative N-glycosylation sites was glycosylated, and the mutation to Ala to avoid glycosylation did not affect the ligand binding affinity. However, the N-terminal region of the mutant was digested at the carboxyl ends of Arg3 and Arg8, as detected by N-terminal amino acid sequencing, and Ser309 in the C-terminal region was partially phosphorylated, as identified in the micro-sequencing by Q-TOF-MS/MS. To avoid chemical heterogeneity, the N-terminal peptide (1-14) truncated and the C-terminal phosphorylation-site eliminated mutant was generated. The binding affinity of the mutant's membrane fraction for LTB(4) was K(d)=6.6 nM and B(max)=50.0 pmol/mg membrane protein. The yield of purified mutant was approximately 0.3-0.4 mg from 1L culture, and the protein showed a single peak at molecular weight of 100 kDa in gel-filtration and no glycosylation or phosphorylation in MALDI-TOF MS.