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N Eguchi

Publications and source records attributed to N Eguchi.

At least 37 records · Page 2Linked to original sources
PubMed1999

Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice.

Prostaglandin (PG) D2 is the most abundant prostanoid produced in the central nervous system of mammals and has been implicated in the modulation of neural functions such as sleep induction, nociception, regulation of body temperature, and odor responses. We generated gene-knockout mice for lipocalin-type PGD2 synthase (L-PGDS) and found that the intrathecal administration of PGE2, an endogenous pain-producing substance, failed to elicit allodynia (touch-evoked pain), which is one typical phenomenon of neuropathic pain, whereas it evoked thermal hyperalgesia, in L-PGDS-/- mice. We also found that the allodynic response induced by the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline was selectively abolished in the L-PGDS-/- mice, among excitatory and inhibitory agents that induced allodynia in wild-type mice. Interestingly, simultaneous injection of a femtogram amount of PGD2 with PGE2 or bicuculline induced allodynia in L-PGDS-/- mice to the same extent as in wild-type mice. The PGE2- or bicuculline-evoked allodynia in wild-type and in PGD2-supplemented L-PGDS-/- mice was blocked by a PGD2 receptor antagonist given in a femtogram amount. These results reveal that endogenous PGD2 is essential for both PGE2- and bicuculline-induced allodynia.

Anesthesia↗
PubMed1999

An apoptotic depletion of oligodendrocytes in the twitcher, a murine model of globoid cell leukodystrophy.

Morphological alterations of oligodendrocytes (OLs) leading to their depletion were studied in the genetic demyelinating mutant, twitcher, a murine model of globoid cell leukodystrophy (GLD). With pi-glutathione-S-transferase immunostaining, OLs with multiple varicose processes were recognized in the early stages and adjacent areas of demyelination and then the OLs cytoplasm as well as the processes became shrunken with progression of the disease. These shrunken OLs were labeled by the TUNEL method, indicative of apoptotic cell death. The ultrastructural features of apoptotic cells were noted in these OLs and DNA laddering was noted in the twitcher brain in advanced stages. This is the first report describing the gradual depletion of OLs by apoptosis in genetic demyelination.

Animals↗
PubMed1999

Gene mutations of K-ras in gallbladder mucosae and gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct.

OBJECTIVE: In this study, we examined the mutational spectrum of K-ras in cases of gallbladder and gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct (AJPBD). METHODS: We examined 35 gallbladders with AJPBD (20 with hyperplasia, 15 with carcinoma) and 38 gallbladders without AJPBD (four normal gallbladders, four with hyperplasia, six with adenoma, 24 with carcinoma). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to detect mutations in codon 12 or 13 of K-ras. RESULTS: In the cases with AJPBD, the prevalences of K-ras mutation were 15% (3/20) in hyperplasia, 60% (6/10) in stage I carcinoma, and 100% (5/5) in stage II-IV carcinoma. In the cases without AJPBD, the prevalences of K-ras mutation were 0% (0/4) in normal gallbladder, 0% (0/4) in hyperplasia, 17% (1/6) in adenoma, 7% (1/16) in stage I carcinoma, and 38% (3/8) in stage II-IV carcinoma. Prevalences of K-ras mutation in hyperplasia and carcinoma with AJPBD were greater than those without AJPBD (p < 0.05). The point mutation of GGT to GAT in codon 12 was frequently observed in the cases with AJPBD. CONCLUSION: These results suggest that the specific K-ras mutation in codon 12 (GGT to GAT) may contribute to the early stage of carcinogenesis in the gallbladder with AJPBD.

Adenoma↗
PubMed1999

Leukocyte differential analysis in multiple laboratory species by a laser multi-angle polarized light scattering separation method.

Leukocyte differential analysis was performed in various species, particularly laboratory animals, by the laser multi-angle polarized light scattering separation method. Venous blood specimens were drawn from the following subjects: healthy adult men and women ("humans"); cynomolgus monkeys ("monkeys"); common marmosets ("marmosets"); beagle dogs ("dogs"); miniature potbelly pigs ("swine"); Japanese white rabbits ("rabbits"); Hartley guinea pigs ("guinea pigs"); and Sprague-Dawley rats ("rats"). 90 degrees/10 degrees scatter plot: Basophils and mononuclear-polymorphonuclear cells were separated in all subjects, but individual 10 degrees and 90 degrees scatter plots overlapped in dogs and guinea pigs, respectively. 90 degrees depolarized /90 degrees scatter plot: Neutrophils and eosinophils were clearly separated in human, monkey, guinea pig, swine and rat subjects. The eosinophil cluster was not clearly plotted in marmoset, dog, or rabbit. 0 degree/10 degrees scatter plot: Regarding this plot for monocytes and lymphocytes, cells were plotted in the following order in all subjects: lymphocytes < basophils < or = monocytes in the 0 degree (size) scatter; and lymphocytes [symbol: see text] monocytes < or = basophils in the 10 degrees (complexity) scatter. Compared to other species, the rat scatter showed a tendency to overlapping plots in both the 0 degree and 10 degrees scatters in the monocyte and lymphocyte clusters. In both dog and guinea pig, the monocyte and neutrophil plots overlapped in the 0 degree and 10 degrees scatters. Basobox: In the human and rabbit subjects, the basophil cluster was plotted within the established basobox, but no clear cell cluster was plotted in the other subjects. As a result of comparing the percentage values for leukocytes in various species obtained by using the CD3500 apparatus versus the corresponding values obtained manually, good correspondence was found in the monkey, and eosinophils in the marmoset were lower with CD3500 than manually. In the rabbit, the mean measured value for basophils matched in the manual and CD3500 findings. In the guinea pig, the CD3500 values were lower than the manual values for lymphocytes, but higher for monocytes and neutrophils. The above findings suggest that the laser multi-angle polarized light scattering separation method is indeed capable of analyzing leukocytes from various species based on cell size and cell complexity, i.e., the presence or absence of nuclei, granules and cell enclosures.

Adult↗
PubMed1998

Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans.

Plasmodium falciparum causes the most severe form of human malaria, which kills approximately 1.5-2.7 million people every year, but the molecular mechanisms underlying the clinical symptoms and the host-parasite interaction remain unclear. We show here that P. falciparum produces prostaglandins (PGs) D2, E2, and F2alpha. After incubation with 1 mM arachidonic acid (AA), cell homogenates of P. falciparum produced PGs as determined by enzyme immunoassay and gas chromatography-selected ion monitoring. PG production in the parasite homogenate was not affected by the nonsteroidal antiinflammatory drugs aspirin and indomethacin, and was partially heat resistant, whereas PG biosynthesis by mammalian cyclooxygenase was completely inhibited by these chemicals and by heat treatment. Addition of AA to the parasite cell culture markedly increased an ability of the parasite cell homogenate to produce PGs and of parasitized red blood cells to accumulate PGs in the culture medium. PGD2 and PGE2 accumulated in the culture medium at the stages of trophozoites and schizonts more actively than at the ring stage. These findings are the first evidence of the direct involvement of a malaria parasite in the generation of substances that are pyrogenic and injurious to the host defenses. We will discuss a possible contribution of the parasite-produced PGs to pathogenesis and host-parasite interaction of P. falciparum.

Animals↗
PubMed1998

The establishment of a preoperative diagnosis of pancreatic carcinoma using cell specimens from pancreatic duct brushing with special attention to p53 mutations.

BACKGROUND: Previously, the authors reported that 82% of cases of pancreatic carcinoma were positive for p53 in cytologic specimens obtained by selective endoscopic pancreatic duct brushing (SEPB). However, there was an extreme discrepancy between the authors' data of p53 overexpression using cytologic specimens and other reports using surgically resected specimens. In this study, the authors demonstrate that p53 positive cells precisely reflect its gene mutations, and also establish systematic procedures for the preoperative diagnosis of patients with pancreatic carcinoma. METHODS: The authors examined 44 cases of pancreatic carcinoma, 30 cases of chronic pancreatitis, and 9 cases of papillary adenoma. In all cases, pathologic diagnosis was made by surgery or autopsy. The conventional cytology and p53 immunocytology were performed simultaneously in the cell specimens obtained by SEPB. In the cases immunostained for p53, DNA was extracted selectively from p53 immunostained cells using a light microscope. p53 mutations in exons 5 to 8 were examined by direct sequencing. RESULTS: Forty of 44 pancreatic carcinomas (91%) were diagnosed correctly by the methods of conventional cytology associated with p53 immunocytology. p53 mutations were detected in 12 of 14 cases that were positive for p53 (86%). Four of six cases that were inoperable due to massive metastasis or invasion had the mutation at codon 273 (CGT to CAT) in exon 8. CONCLUSIONS: These results suggest that p53 immunocytology reflects its gene mutations precisely, and that the point mutation at codon 273 (CGT to CAT) of p53 may play an important role in the invasive potential and metastasis of pancreatic carcinoma.

Adenocarcinoma↗
PubMed1998

Sodium dodecyl sulfate-capillary gel electrophoretic analysis of molecular mass microheterogeneity of beta-trace protein in cerebrospinal fluid from patients with central nervous system diseases.

Molecular mass (M(r)) microheterogeneity of beta-trace protein (beta TP) in cerebrospinal fluid (CSF) from patients with various neurological disorders was analyzed by sodium dodecyl sulfate capillary gel electrophoresis. Under the conditions employed, beta TP with a M(r) distribution of 23,000-30,000 was roughly separated into two subfractions containing the major peaks with M(r) of 26,000 and 28,500, respectively. The peak area ratios of the two subfractions of the electropherograms varied among the samples examined, and elevation in the total beta TP level in the CSF from patients with organic diseases in the central nervous system (CNS) was often accompanied by changes in the ratios of the subfractions. The quantitative changes in the subfraction level in CSF beta TP are considered to reflect the pathological alterations in the CNS.

Adult↗
PubMed1998

Dominant expression of rat prostanoid DP receptor mRNA in leptomeninges, inner segments of photoreceptor cells, iris epithelium, and ciliary processes.

Prostaglandin (PG) D2 is one of the major prostanoids in the mammalian brain and eye tissues. Its function is mediated by the prostanoid DP receptor, which is specific for PGD2 among the various prostanoids. In this study, we cloned the full-length cDNA for the rat DP receptor and used it for detection of DP receptor mRNA in various rat tissues. Northern blotting and RT-PCR analyses revealed that this DP receptor was expressed most intensely in the eye tissues, moderately in the leptomeninges and oviduct, and weakly in the epididymis. The tissue distribution profile of the mRNA for the rat DP receptor is overlapped with those of hematopoietic and lipocalin-type PGD synthases. Among rat eye tissues, the expression was the highest in the iris. In situ hybridization and in situ RT-PCR revealed DP receptor mRNA to be localized in the epithelium of the iris and ciliary body and in photoreceptor cells of the retina, suggesting the involvement of the receptor in the physiological regulation of intraocular pressure and the vision process. In the brain, DP receptor mRNA was dominantly expressed in the leptomeninges and was not detected in the brain parenchyma including the ventral rostral forebrain, the surface area of which is reportedly involved in sleep induction by PGD2.

Amino Acid Sequence↗
PubMed1998

Lipocalin-type prostaglandin D synthase in human male reproductive organs and seminal plasma.

Prostaglandin D synthase (PGDS) activity was detected in human seminal plasma (0.05-1.83 nmol/min per milligram protein). The enzyme was purified from human seminal plasma by immunoaffinity chromatography and found to be 27 kDa in size and N-glycosylated, similar to PGDS in the cerebrospinal fluid. The N-terminal amino acid sequence of 16 residues of the seminal enzyme, APEAQVSVQPNFQQDK, was identical to that of the cerebrospinal fluid PGDS. Although PGDS activity and the content determined by the immunoassay each highly varied in the seminal plasma, the concentration was significantly (p < 0.001) lower in the oligozoospermic group (2.47 +/- 0.51 microg/ml) than in the normozoospermic group (9.75 +/- 1.49 microg/ml). Prostaglandin (PG) D2 was detected in the seminal plasma (5.00 +/- 0.65 ng/ml) with a positive correlation to the PGDS concentration (p < 0.05). PGD2 was converted to the J series of PGs in the seminal plasma with a half-life of 6.5 h. Northern blot analysis revealed that mRNA for PGDS was expressed in the testis, prostate, and epididymis. Through immunohistochemistry, PGDS was localized in Leydig cells of the testis and in epithelial cells of the prostate and ductus epididymidis.

Adult↗
PubMed1998

Identification of a fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase.

The objective of this study was to characterize a 26-kDa seminal plasma protein previously shown to be prevalent in bulls of high fertility. Spots of this protein, excised and electroeluted from two-dimensional SDS-PAGE gels, were used for N-terminal amino acid sequencing and for preparation of antiserum in rabbits. The N-terminal amino acid sequence (ALQPNFEEDKFLGRWFTSGL) was 75% identical and 100% homologous to lipocalin-type prostaglandin (PG) D synthase isolated from human cerebrospinal fluid (CSF). Western blots of purified 26-kDa protein cross-reacted with polyclonal antibodies against lipocalin-type PGD synthase isolated from rat brain and human CSF. Immunoreactive bands at 26 kDa appeared in Western blots of seminal plasma and cauda epididymal fluid (CEF). A 29-kDa band appeared in blots of rete testis fluid (RTF). PGD synthase activity was detected in seminal plasma, CEF, and RTF. The cDNA for bovine lipocalin-type PGD synthase, isolated by reverse transcription-polymerase chain reaction, contained a coding region of 573 base pairs corresponding to 191 amino acids. The amino acid sequence was 63-80% identical to that of the enzyme of other mammals. These results establish that the 26-kDa fertility-associated protein in bull seminal plasma is lipocalin-type PGD synthase. Although we do not yet know the role of lipocalin-type PGD synthase in the male genital tract, we speculate that this protein may play an important role in both the development and the maturation of sperm.

Amino Acid Sequence↗
PubMed1998

Localization of lipocalin-type prostaglandin D synthase (beta-trace) in iris, ciliary body, and eye fluids.

PURPOSE: Prostaglandin (PG) D synthase is present in neural tissues and cerebrospinal fluid (beta-trace). This enzyme belongs to the lipocalin family which consists of transporter proteins for lipophilic substances in the extracellular space. PGD synthase is found in retinal pigment epithelium, from where it is secreted into the interphotoreceptor matrix. The authors have undertaken the localization of this unique enzyme within the tissues and spaces of the anterior segment of the eye. METHODS: Iris, ciliary body, lens, and aqueous and vitreous humors were collected from adult rats and mice. PGD synthase activity was determined, and the protein was quantified by Western blot analysis and localized immunohistochemically. Finally, in situ hybridization was performed to localize PGD synthase mRNA. RESULTS: PGD synthase was most abundant in the aqueous and vitreous humors. It was less abundant in tissue cytosolic fractions; these fractions had almost 10-fold as much as their corresponding membrane-bound fractions. Lens tissue had the lowest amount observed. PGD synthase was localized to the epithelial cells of the iris and the ciliary body and to the adjacent extracellular chambers, but PGD synthase mRNA was found only within the epithelial cells. Several glycosylated forms of PGD synthase were also detected. CONCLUSIONS: PGD synthase was synthesized within the epithelial cells of the iris and the ciliary body and was then secreted into the aqueous and vitreous humors, where it accumulated as an active enzyme.

Animals↗
PubMed1997

Expression of lipocalin-type prostaglandin D synthase (beta-trace) in human heart and its accumulation in the coronary circulation of angina patients.

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in the central nervous system and male genital organs of various mammals and is secreted as beta-trace into the closed compartment of these tissues separated from the systemic circulation. In this study, we found that the mRNA for the human enzyme was expressed most intensely in the heart among various tissues examined. In human autopsy specimens, the enzyme was localized immunocytochemically in myocardial cells, atrial endocardial cells, and a synthetic phenotype of smooth muscle cells in the arteriosclerotic intima, and accumulated in the atherosclerotic plaque of coronary arteries with severe stenosis. In patients with stable angina (75-99% stenosis), the plasma level of L-PGDS was significantly (P < 0.05) higher in the great cardiac vein (0.694 +/- 0.054 microg/ml, n = 7) than in the coronary artery (0.545 +/- 0.034 microg/ml), as determined by a sandwich enzyme immunoassay. However, the veno-arterial difference in the plasma L-PGDS concentration was not observed in normal subjects without stenosis. After a percutaneous transluminal coronary angioplasty was performed to compress the stenotic atherosclerotic plaques, the L-PGDS concentration in the cardiac vein decreased significantly (P < 0.05) to 0.610 +/- 0.051 microg/ml at 20 min and reached the arterial level within 1 h. These findings suggest that L-PGDS is present in both endocardium and myocardium of normal subjects and the stenotic site of patients with stable angina and is secreted into the coronary circulation.

Angina Pectoris↗
PubMed1997

Cloning and crystal structure of hematopoietic prostaglandin D synthase.

Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.

Animals↗
PubMed1997

Analysis of low-molecular-mass proteins in cerebrospinal fluid by sodium dodecyl sulfate capillary gel electrophoresis.

Proteins of low molecular (M(r)) in human cerebrospinal fluid (CSF) were analyzed by capillary electrophoresis in a sodium dodecyl sulfate-containing polymer solution. Under the conditions employed, peaks of beta 2-microglobulin (beta MG) (M(r): 11,700), gamma-trace protein (12,300), myelin basic protein (18,000), beta-trace protein (beta TP) (23,000 to 30,000) and alpha 1-acid glycoprotein (42,000) were detected on the electropherograms. The concentrations of beta MG and beta TP were determined based on the peak area relative to that of an internal standard, Orange G, which was added at a constant amount as the front marker. It was demonstrated that their levels in CSF change under various pathological conditions in the central nervous system.

Adolescent↗
PubMed1997

Immunocytochemical detection of p53 protein as an adjunct in cytologic diagnosis from pancreatic duct brushings in mucin-producing tumors of the pancreas.

BACKGROUND: In cases of mucin-producing tumor (MPT) of the pancreas, a new clinical entity of pancreatic tumor, it has been reported to be difficult to distinguish adenoma from carcinoma preoperatively. This observation caused the authors to develop a new method of p53 immunocytochemistry using cytologic samples obtained by endoscopic retrograde pancreatic duct brushing (ERPDB). METHODS: Fifteen cases of MPT (9 with carcinoma and 6 with adenoma) were examined. In all cases, histologic diagnosis was determined by surgery or autopsy. A wire with a small brush was pushed into the dilatated pancreatic duct detected by endoscopic retrograde pancreatography (ERP). Specimens obtained by brushing were prepared on slides and subsequently fixed with ethanol. Papanicolaou staining and p53 immunocytochemistry were performed simultaneously. The ability of conventional cytology to distinguish adenoma from carcinoma was compared with that of p53 immunocytochemistry. RESULTS: Five of 9 cases (56%) with carcinoma of MPT were correctly diagnosed by Papanicolaou staining. Two of four cases, classified as benign by Papanicolaou staining, expressed p53 protein. Overall, 7 of 9 cases (78%) with carcinoma MPT were correctly diagnosed by Papanicolaou staining associated with p53 immunocytochemistry. In the cases with adenoma MPT, all cases were classified as benign by Papanicolaou staining and no case positive for p53 protein was encountered. CONCLUSIONS: These results suggest that p53 immunocytochemistry as an adjunct in cytologic diagnosis using ERPDB may contribute to differentiating adenoma from carcinoma MPT of the pancreas preoperatively.

Adenocarcinoma, Mucinous↗
PubMed1997

Lipocalin-type prostaglandin D synthase (beta-trace) is a newly recognized type of retinoid transporter.

Lipocalin-type prostaglandin D synthase is responsible for the biosynthesis of prostaglandin D2 in the central nervous system and the genital organs and is secreted into the cerebrospinal fluid and the seminal plasma as beta-trace. Here we analyzed retinoids binding of the enzyme by monitoring the fluorescence quenching of an intrinsic tryptophan residue, and appearance of circular dichroism around 330 nm, and a red shift of the UV absorption spectra of retinoids. We found that the enzyme binds all-trans- or 9-cis-retinoic acid and all-trans- or 13-cis-retinal, but not all-trans-retinol, with affinities (Kd of 70-80 nM) sufficient for function as a retinoid transporter. All-trans-retinoic acid inhibited the enzyme activity in a noncompetitive manner, suggesting that it binds to the same hydrophobic pocket as prostaglandin H2, the substrate for prostaglandin D synthase, but at a different site in this pocket. It is likely that this enzyme is a bifunctional protein that acts as both retinoid transporter and prostaglandin D2-producing enzyme.

Alanine↗