[Thyroid crises and myxedema crises].
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Emergencies↗
Biomedical subjects
K Kitajima
Publications and source records attributed to K Kitajima.
Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis.
Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i). Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+). N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199-27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.
Adult↗
Elevated expression of free deaminoneuraminic acid in mammalian cells cultured in mannose-rich media.
Deaminoneuraminic acid (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is a member of the family of sialic acids in which an acylamino group at the C-5 position of N-acylneuraminic acid (Neu5Acyl) is replaced by a hydroxyl group. It has recently been shown that KDN is synthesized de novo from its precursor, mannose (Man), in trout testis (Angata, T., Nakata, D., Matsuda, T., Kitajima, K., and Troy, F. A. (1999) J. Biol. Chem. 274, in press). In this study, we examined the effect of extracellular free Man on biosynthesis of KDN in mouse melanoma B16 and African green monkey kidney COS-7 cell lines. The following new findings are reported. First, the levels of free and bound forms of KDN increased when the cells were cultured in the presence of 20 mM Man. The level of intracellular free KDN in COS-7 and B16 cells increased 47- and 66-fold respectively, compared with the levels in control cells. Second, the elevated expression of free KDN was proportional to the intracellular concentration of free Man. Third, KDN 9-phosphate (KDN-9-P) synthase, which condenses Man 6-phosphate and phosphoenolpyruvate (PEP), forming KDN-9-P, was detected in cell lysates from both cell lines. Fourth, the de novo synthesis of KDN in both cell lines in the Man-rich media was unaffected by the addition of N-acetylmannosamine (ManNAc), the hexosamine precursor for synthesis of N-acetylneuraminic acid (Neu5Ac). These results show that KDN is synthesized using free Man as its hexose precursor in these mammalian cells. Thus, the KDN biosynthetic pathway utilizes enzymes distinct, at least in part, from those involved in Neu5Ac biosynthesis. This is the first report showing that in vivo synthesis of KDN can be manipulated by growing cells in the presence of Man. This now provides a useful method to study the metabolism and function of the KDN glycotope.
Animals↗
Cholesterol-dependent localization of NAP-22 on a neuronal membrane microdomain (raft).
A membrane microdomain called raft has been under extensive study since the assembly of various signal-transducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl-beta-cyclodextrin was found to be effective to solubilize NAP-22, a neuron-enriched Ca(2+)-dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dose-dependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.
Animals↗
Isolation and characterization of low density detergent-insoluble membrane (LD-DIM) fraction from sea urchin sperm.
The low density detergent-insoluble membrane (LD-DIM) fraction was obtained by a sucrose-density gradient centrifugation from sperm of three sea urchin species, Hemicentrotus pulcherrimus, Strongylocentrotus purpuratus, and Anthocidaris crassispina. These LD-DIM preparations were characterized by enriched glycosphingolipids (GSL) including gangliosides and sulfatide (SLF), having more than 50% of the total amount of GSL present in these sperm. Interestingly, a minor component of H. pulcherrimus sperm (HO3S-->8Neu5Acalpha2-->8Neu5Acalpha2-->6Glcbeta1++ +-->Cer) was shown to be even more enriched in the LD-DIM as revealed by using monoclonal antibody (mAb.3G9) speific to this ganglioside. In addition to the GSL, phosphatidyl-serine (PS) and diacylglcerol (DG) were enriched in the LD-DIM. On the other hand, cholesterol (CL) and sphingomyelin (SM) were not so enriched, which contrasted with the LD-DIM from Madin-Darby canine kidney (MDCK) cells, where CL and SM were reported to be abundant. Because mammalian somatic cell-derived DIMs have been proposed to be associated with functional signal transduction, it seems possible that the ganglioside-enriched LD-DIM in sea urchin sperm can participate in binding to eggs and the subsequent egg activation process. To our knowledge this is the chemical characterization of the LD-DIM fraction of a gametic cell.
Animals↗
Regulation of inner ear fluid in the rat by vasopressin.
The anti-diuretic hormone vasopressin has been shown to be important in regulating inner ear fluid. The diuretic hormone, CNP, and its receptor, ANP-B receptor, may also function in the regulation of inner ear fluid. To determine whether vasopressin directly affects the fluid level, we infused this hormone to rat and assay of V2-AVP receptor mRNA by semiquantitative RT-PCR demonstrated a significantly lower level of this transcript in vasopressin-infused animals than in saline-infused animals. The levels of CNP and ANP-B receptors mRNA, however, were the same in both groups of rats. Results suggest that high plasma levels of vasopressin may be a principal causal factor of endolymphatic hydrops in Meniere's disease, perhaps by down-regulating the number of vasopressin receptors.
Animals↗
Lactation-dependent expression of an mRNA splice variant with an exon for a multiply O-glycosylated domain of mouse milk fat globule glycoprotein MFG-E8.
Expression of mRNA encoding MFG-E8, a milk fat-associated glycoprotein was investigated in mouse mammary gland. Two forms of mRNA, long and short variants, were shown to be expressed in the mammary tissue by RT-PCR analysis. Sequence analyses of these two variants and an isolated MFG-E8 gene segment indicated that the long and short mRNA variants resulted from an alternative splicing of a single pre-mRNA through in-flame inclusion and skipping of one exon, which encodes a proline/threonine (Pro/Thr)-rich domain. The long variant was expressed predominantly in mammary gland and the expression level was remarkably increased at late gestation and kept high during lactation. On the contrary, the short variant was detected ubiquitously in various tissues and its expression in the mammary gland was rather decreased in a lactation dependent manner. Expression of the long variant was also detected in a mouse mammary epithelial cell line, COMMA-1D, and enhanced by incubation with lactogenic hormones. Glycosylation inhibition analyses using tunicamycin and alpha-benzyl-GalNAc were conducted with COS7 cells transfected with plasmids expressing each mRNA variant, demonstrating that a fully glycosylated product of the long mRNA variant was not only N-glycosylated but also multiply O-glycosylated, whereas a product of the short one had only N-glycan(s). These results suggest that the alternative splicing plays a critical role for the mammary-specific and lactation-dependent expression of the MFG-E8 isoform and that the multiply O-glycosylated Pro/Thr-rich domain of this isoform is functionally important for formation of milk fat globules in mammary epithelial cells.
Alternative Splicing↗
Fluorescent-assisted detection of oligosialyl units in glycoconjugates.
A highly sensitive chemical method to detect various types of oligo/polysialic acid (oligo/polySia) units in glycoconjugates, i.e., alpha2 --> 8-linked homo-oligo/polySia [N-acetylneuraminic acid (Neu5Ac), N-glcolylneuraminic acid (Neu5Gc), or 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid], alpha2 --> 8-linked heterodimers of Neu5Ac and Neu5Gc, alpha2 --> 9-linked homooligo/polyNeu5Ac, and alpha2 --> 5-Oglycolyl-linked homooligo/polyNeu5Gc, was developed with an alpha-keto acid-reactive fluorescent labeling reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB). Fluorescent labeled di- or oligoSia was separated and quantitated by fluorometric anion-exchange high-performance liquid chromatography (HPLC). As little as 13 fmol of Neu5Acalpha2 --> 8Neu5Ac was detectable by this method. When alpha2 --> 8-linked oligo/polyNeu5Ac with on average eight Neu5Ac residues was labeled with DMB, DMB derivatives of oligomers with only lower degrees of polymerization of 2 to 7, were detected, due to concomitant partial depolymerization of oligo/polySia chain with the derivatization. For glycoproteins and glycolipids, mild acid hydrolysis was performed to release oligoSia prior to DMB derivatization. The mild acid hydrolysis/fluorometric HPLC method was also applicable to glycoprotein samples blotted on the membrane.
Animals↗
Definitive but not primitive hematopoiesis is impaired in jumonji mutant mice.
A novel gene, jumonji was identified by a mouse gene trap strategy. The jumonji gene encodes a protein containing a putative DNA binding domain. The mice homozygous for jumonji gene with a BALB/cA genetic background show hypoplasia of the fetal liver and embryonic lethality, suggesting impaired hematopoiesis. In the peripheral blood of jumonji mutant embryos, the number of fetal liver-derived definitive erythrocytes, but not yolk sac-derived primitive erythrocytes, showed a marked reduction, suggesting that jumonji mutants die of anemia. The defects of definitive erythrocytes in jumonji mutants seemed to be caused by a decrease in the numbers of multiple hematopoietic progenitors including colony-forming unit-spleen (CFU-S) in the fetal liver. However, hematopoietic stem cells (HSCs) in the fetal liver of jumonji mutants could reconstitute the hematopoietic system of lethally irradiated recipients. In the fetal liver, the jumonji gene is expressed in fibroblastic cells and endothelial cells, but not in Lin-/c-Kit+/Sca-1(+) cells known to include HSCs. These results suggest that an environmental defect induce the impaired hematopoiesis in the fetal liver of jumonji mutant embryos.
Animals↗
Expression of a novel type of classic cadherin, PB-cadherin in developing brain and limb buds.
PB-cadherin is a novel classic type of cadherin predominantly expressed in brain of adult rats (Sugimoto et al. [1996] J. Biol. Chem. 271:11548-11556). To examine the spatial and temporal expression of PB-cadherin during development, we isolated full-length cDNA of mouse PB-cadherin and studied its expression pattern in mouse embryos. In Northern blots, PB-cadherin mRNA was detected at 9.5 days postcoitum (dpc) onwards. Whole-mount in situ hybridization showed that PB-cadherin signals mainly occurred in developing neural tissues, including brain and spinal cord, and limb buds in the 10.5 dpc embryo. In the brain, PB-cadherin mRNA were strongly expressed in the forebrain and midbrain-hindbrain boundary region (isthmus). In isthmus, PB-cadherin expression delineated the expression area of Wnt-1, a secreted signaling molecule essential for proper cerebellum development. In the developing limb, PB-cadherin mRNA was first localized in posterior part of buds at 10.5 dpc, and was thereafter distributed in a domain around the digit rudiments. This expression pattern is similar to that of BMP-2, a secreted signalling molecule involving limb patterning and morphogenesis. These findings suggested the possibility that PB-cadherin-mediated cell-cell adhesion has a functional role in pattern formation and morphogenesis of mouse embryonic brain and limb. Dev Dyn 1999;215:206-214.
Amino Acid Sequence↗
Culture of human parathyroid cells for transplantation.
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Antigens, CD↗
Immunohistochemical localization of choline acetyltransferase of a peripheral type in the rat larynx.
As shown in the accompanying paper, choline acetyltransferase, so far the best histochemical marker for identifying cholinergic structures, has at least one alternative splice variant. The variant, termed pChAT because of its preferential expression in peripheral organs, encouraged us to study peripheral, probably cholinergic, cells and fibers by immunohistochemistry using an antiserum against a peptide specific for pChAT. We chose the larynx of the rat, since cholinergic innervation in this organ has been well established by physiological studies, but not sufficiently by chemical neuroanatomy. Neuronal somata positive for pChAT were found in the intralaryngeal ganglia. Our double staining study indicated that these somata always possessed acetylcholinesterase activity, while the reverse did not hold true. Nerve fibers positive for pChAT were distributed widely in the intrinsic laryngeal muscles, laryngeal glands, blood vessels and laryngeal mucosa. In the intrinsic laryngeal muscles, pChAT-positive terminals were apposed closely to motor end-plates which were stained positively for acetylcholinesterase activity. Denervation experiments revealed that there were three types of pChAT-positive fibers in the larynx: (1) special visceral efferent fibers to the intrinsic laryngeal muscles, which decreased dramatically in number after vagotomy; (2) parasympathetic postganglionic fibers near the laryngeal glands and blood vessels, which appeared unaffected after vagotomy or cervical sympathectomy: and (3) afferent fibers innervating the laryngeal mucosa, which reduced markedly in number after vagotomy performed distal, but not proximal, to the nodose ganglion. Such afferent fibers remained unchanged following the neonatal capsaicin treatment, suggesting their independence from those containing substance P.
Afferent Pathways↗
Successful cytokine treatment of aplastic anemia following living-related orthotopic liver transplantation for non-A, non-B, non-C hepatitis.
The relationship between aplastic anemia and viral hepatitis is well recognized, and such patients usually have a high mortality. We successfully treated a case of aplastic anemia following living-related orthotopic liver transplantation (LROLT) for non-A, non-B, non-C hepatitis. A 2-yr-old boy with fulminant hepatic failure from non-A, non-B, non-C hepatitis received LROLT. Before transplantation, he had pancytopenia which was probably hepatitis associated, and viral suppression was suspected after bone marrow (BM) biopsy. After the transplantation, he developed progressive pancytopenia and a diagnosis of aplastic anemia was made via BM biopsy. With immunosuppressant agents (cyclosporine, methylprednisolone), cytokine therapy (granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), recombinant human erythropoietin (rhEPO)) was effectual and the patient recovered from pancytopenia. He was discharged from the hospital 57 d after the liver transplantation and remains well 1 yr after LROLT. Combined cytokine therapy with high doses of G-CSF, M-CSF and rhEPO appeared to be effective in the treatment of aplastic anemia following liver transplantation for non-A, non-B, non-C hepatitis. Since M-CSF activates macrophages, it may have contributed to the graft rejection. Careful consideration should be given to the use of high-dose M-CSF in liver transplant patients.
Anemia, Aplastic↗
A 42-kDa glycoprotein from chicken egg-envelope, an avian homolog of the ZPC family glycoproteins in mammalian Zona pellucida. Its first identification, cDNA cloning and granulosa cell-specific expression.
A glycoprotein with molecular mass of 42 kDa was identified as the major component of the chicken egg-envelope, the filamentous, extracellular matrix known as the perivitelline layer. By using a DNA probe amplified with degenerative primers derived from the protein's partial amino acid sequences, a cDNA clone encoding the egg-envelope 42-kDa glycoprotein (gp42) was isolated from a hen's ovary cDNA library. The gp42 open reading frame encoded 435 amino acid residues, including a putative signal peptide of 20 amino acids. The deduced amino acid sequence of gp42 showed significant similarity to egg-envelope glycoproteins of the ZPC family of several other vertebrate species, including human ZP3, mouse ZP3, Xenopus laevis gp43 and medaka (Oryzias latipes) ZI3 (LS-F), which play important roles for sperm-egg interaction. A single N-glycosylation site present in chicken gp42 is conserved among all five of these proteins: carbohydrate analysis of gp42 revealed the presence of a complex type glycan chain at this site. N-terminal sequence analysis of the mature polypeptide suggests that C-terminal processing of the pro-protein occurs during synthesis and secretion. The 1.4-kb gp42 transcript was detected only in follicles, and was found to be accumulated in granulosa cells in a manner dependent on ovarian follicular development. Furthermore, a metabolically radio-labeled gp42 was immunopreciptated from both cell lysate and culture supernatant of the granulosa cells with specific anti-gp42 antibody, suggesting granulosa cell-specific synthesis and secretion of the glycoprotein.
Amino Acid Sequence↗
Expression of inducible nitric oxide synthase in human thyroid papillary carcinomas.
To evaluate the relationship between nitric oxide (NO) and carcinogenesis, we assayed 4 human thyroid papillary carcinomas (TPC) and 3 normal thyroid glands for the presence of inducible nitric oxide synthase (iNOS). Using an antibody against iNOS, we observed immunohistochemical staining if iNOS in the TPC samples, but not in normal thyroid. When we incubated TPC samples with antibodies against both iNOS and human leukocyte antigen (LCA), a macrophage marker, we found that while most TPC cells were stained with anti-iNOS antibody, only a few were stained with both. Reverse transcription polymerase chain reaction (RT-PCR) showed that iNOS mRNA was expressed in the samples of TPC, but not in normal thyroid. The sequence of iNOS message in the TPC samples was identical to that previously detected in a human colon cancer cell line. These results suggest that iNOS in human TPC is mostly derived from tumor cells, rather than macrophages, and that it may play a direct role in carcinogenesis.
Base Sequence↗
Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.
Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.
Amidohydrolases↗
Comparative study of vocal function after near-total laryngectomy.
OBJECTIVES: To assess vocal function after near-total laryngectomy (NTL) and to compare that with after vertical hemilaryngectomy (VHL), both of which use the laryngeal mucosa as the vibratory tissue. STUDY DESIGN: We retrospectively evaluated 29 patients, 11 of whom underwent NTL and 18 of whom underwent VHL. However, 2 patients failed to speak and 1 patient's voice sample was lost following NTL, so we analyzed the voices of 8 patients who underwent NTL and 18 patients who underwent VHL. METHODS: Vocal function was examined by acoustic and aerodynamic analysis. Acoustic analysis involved the evaluation of pitch, intensity, pitch perturbation quotient, amplitude perturbation quotient, and normalized noise energy. In aerodynamic analysis, we studied the mean airflow rate, vocal efficiency index (alternating current/direct current [AC/DC]), and maximum phonation time. RESULTS: Vocal function except pitch after either NTL or VHL had much variability, but the results following each procedure did not differ significantly. CONCLUSIONS: There are no significant differences between vocal function following NTL and VHL. Fiberscopic examination of the mucosa of the arytenoid was observed to vibrate in patients who underwent NTL, so that the phonatory mechanism after NTL resembled that after VHL.
Adult↗
Peptides glycosylated in the endoplasmic reticulum of yeast are subsequently deglycosylated by a soluble peptide: N-glycanase activity.
Several lines of evidence suggest that soluble peptide:N-glycanase (PNGase) is involved in the quality control system for newly synthesized glycoproteins in mammalian cells. Here we report the occurrence of a soluble PNGase activity in Saccharomyces cerevisiae. The enzyme, which was recovered in the cytosolic fraction, has a neutral pH optimum, and dithiothreitol is required for activity. All of these properties were similar to those of earlier described for mammalian PNGases. Interestingly, the yeast enzyme activity was found to be present almost exclusively in cells in stationary phase; little activity was detected in logarithmic growth phase cells. Upon incubation of a glycosylatable peptide R-Asn-X-Thr-R' with permeabilized yeast spheroplasts, we detected formation of both glycosylated peptide and the peptide product expected from PNGase-mediated deglycosylation of this glycopeptide, namely, R-Asp-X-Thr-R'. Recent findings that yeast have an active system for the retrograde transport of unfolded (glyco)proteins and glycopeptides out of the endoplasmic reticulum (ER) into the cytosol raise the possibility that this PNGase may participate in an early step in degradation of these molecules following their export from the ER.
Amidohydrolases↗