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Biomedical subjects

K Kitajima

Publications and source records attributed to K Kitajima.

At least 55 records · Page 3Linked to original sources
PubMed2000

Endoscopic surgery for a parathyroid functioning adenoma resection with the neck region-lifting method.

Recently, endoscopic surgery has been applied to cervical exploration. We have developed new techniques for endoscopic neck surgery. We reported on a 53-year-old Japanese man with functioning parathyroid adenoma resected by endoscopic surgery with a neck region-lifting method. A 10-mm midline trocar for the endoscope and two 5-mm lateral trocars were inserted from the anterior chest wall to avoid neck scars. There were no intraoperative complications. The incisions were completely covered by the patient's undergarments.

Adenoma↗
PubMed2000

An ELISA-based assay for detergent-solubilized cellular beta 1,4-galactosyltransferase activity. Use of a polyacrylamide derivative with GlcNAc-beta side chains as a solid phase acceptor substrate.

In our previous paper [Oubihi et al. (1998) Anal. Biochem., 257, 169-175], we have shown that a polyacrylamide-derived synthetic glycopolymer with GlcNAcbeta side chains, termed PAP(GlcNAcbeta), is useful as a solid phase acceptor substrate for the ELISA-based analyses of soluble beta1,4(-)galactosyltransferase (GalT) activity in milk. This method is now used to assay detergent-solubilized cellular GalT. The glycopolymer coated on polystyrene plates was shown to be highly stable against the non-ionic and ionic detergents tested (0 approximately 5% solutions of Triton X-100 and SDS). Such stability made it possible to incubate the ELISA plate with detergent-solubilized GalT and to wash the ELISA plate with SDS solution after the GalT reaction, leading to high accuracy and sensitivity of this assay. The GalT activity was assayed using this method for 1% Triton X-100 extracts of various tissue samples of mice and several cultured cell lines. The results showed that the specific GalT activity of tissue extracts was low in brain and intestine, and high in ovary, muscle, and kidney. As for the cultured cell lines, COS7, COMMA-1D and C2C12 cells showed high specific activity, while CHO and MDCK cells showed low activity. The myoblast C2C12 had a slight increase in GalT activity during starvation-induced cell differentiation. On the other hand, GaIT-I transcript estimated by RT-PCR rather decreased during C2C12 cell differentiation, suggesting a differentiation-dependent switch in GalT isozymes. Taken all together, the ELISA-based assay using PAP(GlcNAcbeta) as a solid phase acceptor substrate was demonstrated to be a useful method for the assay of membrane-bound galactosyltransferases.

Acrylic Resins↗
PubMed2000

[Auditory cortical response to monaural stimulation as detected by functional magnetic resonance imaging].

In order to confirm the crossed-innervation between auditory cortex and the ear that receives monosyllabic sound, the auditory cortical response to monaural monosyllabic stimulation as detected by functional magnetic resonance imaging (fMRI) was investigated in six normal hearing subjects. Stimulus amplitude averaged 95 dBSPL at the distal end of the audio system. A series of 440 echo planar images was acquired during the acoustic stimulation within the four OFF-ON cycle paradigm. Five image series with 10 slices were collected within each OFF or On period. Each scanning session began with four baseline images before the OFF-ON paradigm. Monosyllabic sounds were presented monaurally during the ON period at a rate of one monosyllable/sec. Functional MRI data were analyzed with SPM99b software (Statistical Parametric Mapping). The background scanner noise averaged 97dBSPL. The selicon ear plug and headphone as acoustic shields attenuated the noise as much as 17 dB. A broad and intense auditory cortical response was observed bilaterally in response to monaural monosyllable stimulation. Sound presentation to the right ear was followed by a larger response in the left auditory cortex than in the right, and left ear stimulation evoked a larger response in the right auditory cortex than in the left. This pattern was consistent in all subjects examined. The primary auditory cortex responded to monosyllabic words presented to the contralateral ear. The results confirmed the crossed-innervation between the auditory cortex and ear for listening to monosyllables. Functional MRI is a useful tool for investigating auditory cortex function, if the scanner noise is adequated controlled.

Acoustic Stimulation↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

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↗