Pancreatic hamartoma: Possibility of a preoperative diagnosis via endoscopic ultrasound-guided fine-needle aspiration biopsy.

Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) is useful for preoperatively diagnosing various pancreatic tumors. Although there is a risk of complications, such as pancreatitis, this procedure achieves the crucial need of reducing unnecessary invasive surgery for benign lesions. Herein, we reported a surgically resected case of pancreatic hamartoma in the pancreatic head whose retrospective analysis revealed that the specimens obtained via EUS-FNAB contained hamartoma fragments. Pancreatic hamartoma is an extremely rare benign disease that is exceptionally difficult to diagnose before surgical resection owing to its rarity and lack of established imaging findings. To the best of our knowledge, the preoperative diagnosis of pancreatic hamartoma via EUS-FNAB specimens has not been reported to date. Herein, postoperative EUS-FNAB evaluation revealed a collection of pancreatic hamartoma lesions, although the initial diagnosis was pancreatic tissue with focal atrophy and fibrosis. Diagnosis using EUS-FNAB can be challenging owing to the very small sample size. If mature acini and ducts with fibrous stroma without islets are observed in the EUS-FNAB specimen, pancreatic hamartoma should be considered as a differential diagnosis. Thus, careful follow-up or reexamination of EUS-FNAB should be considered instead of surgery if a benign lesion is suspected preoperatively.

Aspartate racemase and D-aspartate in starfish; possible involvement in testicular maturation.

D-Aspartate, aspartate racemase activity, and D-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectinifera was significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index ≥6% was four-fold higher than that of individuals with a gonad index <6%. The ratio of the D-form of aspartate to total aspartate was approximately 25% in testes with a gonad index <6% and this increased to approximately 40% in testes with a gonad index ≥6%. However, such changes were not observed in female ovaries. Administration of D-aspartate into male starfish caused testicular growth. These results indicate the possible involvement of aspartate racemase and D-aspartate in testicular maturation in echinoderm starfish.

Heterologous expression of Halomonas halodenitrificans nitric oxide reductase and its N-terminally truncated NorC subunit in Escherichia coli.

Halomonas halodenitrificans nitric oxide reductase (NOR) is the membrane-bound heterodimer complex of NorC, which contains a low-spin heme c center, and NorB, which contains a low-spin heme b center, a high-spin heme b3 center, and a non-heme FeB center. The soluble domain of NorC, NorC* (ΔMet1-Val37) was heterologously expressed in Escherichia coli using expression plasmids harboring the truncated norC gene deleted of its 84 5'-terminal nucleotides. Analogous scission of the N-terminal helix as the membrane anchor took place when the whole norC gene was used. NorC* exhibited spectra typical of a low-spin heme c. In addition, NorC* functioned as the acceptor of an electron from a cytochrome c isolated from the periplasm of H. halodenitrificans and small reducing reagents. The redox potential of NorC* shifted ca. 40mV in the negative direction from that of NorC. Unlike NorC, recombinant NorB was not heterologously expressed. However, recombinant NOR (rNOR) could be expressed in E. coli by using a plasmid harboring all genes in the nor operon, norCBQDX, from which the three hairpin loops (mRNA) were deleted, and by using the ccm genes for the maturation of C-type heme. rNOR exhibited the same spectroscopic properties and reactivity to NO and O2 as NOR, although its enzymatic activity toward NO was considerably decreased. These results on the expression of rNOR and NorC* will allow us to develop more profound studies on the properties of the four Fe centers and the reaction mechanism of NOR from this halophilic denitrifying bacterium.

Regulation of secondary follicle growth by theca cells and insulin-like growth factor 1.

Ovaries contain follicles at various stages of development, including primordial, primary, secondary, antral and Graafian follicles. Although the growth of these follicles is controlled to maintain regular ovulation, the mechanism through which this occurs remains unclear. In our study, we found that the growth rate of cultured secondary follicles separated from mice ovaries differed between follicles. After 4 days of culture, the size of some secondary follicles was markedly increased, while that of others had either slightly increased, remained unchanged or shrunk. We compared the expression levels of growth factors between these secondary follicles and found that the growth rate of cultured secondary follicles correlated with the expression level of insulin-like growth factor 1 (Igf1) mRNA. Igf1 mRNA expression level in secondary follicles containing theca cells was higher than that in secondary follicles without theca cells, and the granulosa cell proliferation around follicles containing theca cells was increased. Furthermore, an IGF1 inhibitor also inhibited the granulosa cell proliferation, and administration of IGF1 to secondary follicles without growth promoted granulosa cell proliferation. These results indicated that the theca cells of secondary follicles induced the expression of IGF1 and promoted the follicle growth.

Involvement of heparan sulfate 6-O-sulfation in the regulation of energy metabolism and the alteration of thyroid hormone levels in male mice.

Here, we report that male heparan sulfate 6-O-sulfotransferase-2 (Hs6st2) knockout mice showed increased body weight in an age-dependent manner even when fed with a normal diet and showed a phenotype of impaired glucose metabolism and insulin resistance. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of mitochondrial uncoupling proteins Ucp1 and Ucp3 was reduced in the interscapular brown adipose tissue (BAT) of male Hs6st2 knockout mice, suggesting reduced energy metabolism. The serum level of thyroid-stimulating hormone was significantly higher and that of thyroxine was lower in the knockout mice. When cultures of brown adipocytes from wild-type and Hs6st2 knockout mice isolated and differentiated in vitro were treated with FGF19 (fibroblast growth factor 19) or FGF21 in the presence or the absence of heparitinase I, phosphorylation of p42/p44 mitogen-activated protein (MAP) kinase was reduced. Heparan sulfate (HS) 6-O-sulfation was reduced not only in BAT but also in the thyroid tissue of the knockout mice. Thus, 6-O-sulfation in HS seems to play an important role in mediating energy metabolism by controlling thyroid hormone levels and signals from the FGF19 subfamily proteins, and the alteration of the HS composition may result in metabolic syndrome phenotypes such as altered glucose and insulin tolerance.

Determination of D-aspartate N-methyltransferase activity in the starfish by direct analysis of N-methyl-D-aspartate with high-performance liquid chromatography.

We describe a method for the detection and quantification of D-aspartate N-methyltransferase activity. The enzyme catalyzes the S-adenosyl-L-methionine-dependent N-methylation of D-aspartate to form N-methyl-D-aspartate (NMDA). NMDA is detected directly by high-performance liquid chromatography (HPLC) of their (+)- and/or (-)-1-(9-fluorenyl)ethyl chloroformate fluorescent derivatives. The NMDA production in the assay mixture is linearly proportional to the incubation time and the amount of tissue homogenate. Using a 10 min incubation time, the method allows detection of the enzyme activity below 10 fmol/min. It can be used to analyze kinetic behavior and to quantify the enzyme from a wide variety of organisms.

Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88.

Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O-sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293, and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O, and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes.

Specific inhibition of FGF-2 signaling with 2-O-sulfated octasaccharides of heparan sulfate.

In fibroblast growth factor (FGF)-2 signaling, the formation of a ternary complex of FGF-2, tyrosine-kinase fibroblast growth factor receptor (FGFR)-1, and cell surface heparan sulfate (HS) proteoglycan is known to be critical for the activation of FGFR-1 and downstream signal transduction. Exogenous heparin polymer and some octasaccharides inhibited FGF-2-induced phosphorylation both of FGFR-1 and of extracellular signal-regulated kinase (ERK1/2) in Chinese hamster ovary (CHO)-K1 cells transfected with FGFR-1, which present HS on their cell surface. The inhibitory effect of octasaccharide was dependent on the number of 2-O-sulfate groups within a molecule but independent of the number of 6-O-sulfate groups. Sulfation at the 2-O-position was a prerequisite not only for the binding of HS to FGF-2 but also for regulation of FGF-2 signaling and competitive inhibition with endogenous HS. Interestingly, FGF-4-induced phosphorylation was impeded only by specific octasaccharides containing both 2-O- and 6-O-sulfated groups, which were necessary for binding FGF-4. In CHO-677 cells deficient in HS biosynthesis, heparin enhanced FGF-2-induced phosphorylation of ERK1/2. On the other hand, an FGF-2-binding octasaccharide inhibited the phosphorylation. Our data suggest that the activity of particular heparin-binding factors can be inhibited by distinctive oligosaccharides that can bind the factors but cannot form functional signaling complexes irrespective of whether cells have a normal complement of HS or lack HS.

Exclusion of actin-binding protein p57/coronin-1 from bacteria-containing phagosomes in macrophages infected with Legionella.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a human pathogen that multiplies within alveolar macrophages. L. pneumophila establishes specialized phagosomes in which it evades the host defense through largely unknown mechanisms. Here we analyzed the role of an actin-binding protein, p57/coronin-1, a member of the coronin protein family, during Legionella infection. On fluorescence microscopy, p57/coronin-1 and F-actin were found to be co-localized at the sites on the plasma membrane where L. pneumophila adhered to U937 human macrophage-like cells. The localization of p57/coronin-1 at the sites of bacterial adherence was inhibited by treatment with cytochalasin D (an inhibitor of actin polymerization), suggesting that p57/coronin-1 is involved in the actin-dependent uptake of L. pneumophila into U937 cells. In addition, we showed that p57/coronin-1 was excluded from phagosomes containing live L. pneumophila throughout the infection, whereas transient accumulation of p57/coronin-1 was observed on phagosomes containing Texas-Red-labeled opsonized zymosan (TROpZ) or heat-killed L. pneumophila at an early stage of phagocytosis. The exclusion of p57/coronin-1 from phagosomes containing live another Legionella species Legionella gratiana at an early stage of infection was also observed. Taken together, these results suggest that the endocytic pathways of live Legionella species are distinct from general phagocytic pathways, which lead to lysosomal degradation.

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture.

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.

Mice deficient in heparan sulfate 6-O-sulfotransferase-1 exhibit defective heparan sulfate biosynthesis, abnormal placentation, and late embryonic lethality.

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes due to its ability to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. The divergent structures of HS glycosaminoglycans are the result of the coordinate actions of several N- and O-sulfotransferases, C5-epimerase, and 6-O-endosulfatases. We have shown that 6-O-sulfation of the glucosamine residues in HS are catalyzed by the sulfotransferases HS6ST-1, -2, and -3. To determine the biological and physiological importance of HS6ST-1, we now describe the creation of transgenic mice that lack this sulfotransferase. Most of our HS6ST-1-null mice died between embryonic day 15.5 and the perinatal stage, and those mice that survived were considerably smaller than their wild-type littermates. Some of these HS6ST-1-null mice exhibited development abnormalities, and histochemical and molecular analyses of these mice revealed an approximately 50% reduction in the number of fetal microvessels in the labyrinthine zone of the placenta relative to that in the wild-type mice. Because we observed a modest reduction in VEGF-A mRNA and protein in the tissues of HS6ST-1-null mice, an HS-dependent defect in cytokine signaling probably contributes to increased embryonic lethality and decreased growth. Biochemical studies of the HS chains isolated from various organs of our HS6ST-1-null mice revealed a marked reduction of GlcNAc(6SO(4)) and HexA-GlcNSO(3)(6SO(4)) levels and a reduced ability to bind Wnt2. Thus, despite the presence of three closely related 6-O-sulfotransferase genes in the mouse genome, HS6ST-1 is the primary one used in HS biosynthesis in most tissues.