A monoamine in the gustatory cell of the frog's taste organ

Summary Fluorescence histochemistry reveals that in the frog's taste organ a yellow fluorescence is regularly observed at the most basal region of the sensory epithelium. The fluorescence has a strong intensity, but it fades rapidly upon the UV-irradiation. The peak of the emission spectrum is at 520 m. Following reserpine treatment the yellow fluorescence is markedly reduced, but not depleted completely. From these characteristics the monoamine fluorescence is regarded as representing 5-HT (serotonin).The ultrastructural study on sensory epithelia shows that the terminal portions of gustatory cell processes are localized at the basal region. These portions are filled with dense cored vesicles (700–1000 Å in diameter) and frequently opposed with nerve fibers penetrating into the epithelium. The gustatory cell processes are also interposed between the terminal portions or nerve fibers. The cytoplasm of the gustatory cell process is characterized by many mitochondria, fine filaments and glycogen particles, but contains few cored vesicles. The distribution of terminal portions of gustatory cell processes seems to correspond fairly well to that of the monoamine fluorescence observed discontinuously along the basal lamina. Accordingly it is concluded that the fluorigenic monoamine is localized in the cored vesicles of the gustatory cell.These results were reported in a preliminary form to the October, 1974 meeting of the Japan Society of Histochemistry and Cytochemistry.The authors gratefully acknowledge the support and helpful advice of Prof. Dr. T. Kanaseki.     

Selective cell-surface expression of dipeptidyl peptidase IV with mutations at the active site sequence.

The cell surface expression of dipeptidyl peptidase IV (DPPIV) was examined in COS-1 cells transfected with its cDNA with or without mutations at the active site sequence Gly-Trp-Ser-Tyr-Gly (positions 629-633). Mutants with substitution of Trp630----Glu or Ser631----Ala were expressed on the cell surface as normally as the wild-type DPPIV, although the mutant with Ala631 had no enzyme activity. In contrast, any single substitutions of Gly at positions 629 and 633 resulted in no surface expression of the mutants, which were, instead, detected within the cells. When Tyr632 was substituted, one mutant (Tyr----Phe) was expressed on the surface, whereas the others (Tyr----Gly or Leu) were intracellularly retained. These results indicate that the surface expression of DPPIV is critically influenced by mutations at the active site sequence.     

Molecular cloning of nonspecific cross-reacting antigens in human granulocytes

To clarify the molecular structures of nonspecific cross-reacting antigens (NCAs), a family of glycoproteins antigenically related to carcinoembryonic antigen (CEA), in human granulocytes, we have screened a cDNA library of human leukocytes using a cDNA probe for the N-terminal domain (domain-N) of NCA-50, an NCA species in tumor cells. In 95 positive clones randomly selected, we identified six NCA or NCA-related cDNA clones including NCA-50, biliary glycoprotein protein a, and W272 (CGM6) which have previously been reported, and three new clones, W236, W264, and W282, encoding three novel NCA species. W236 and W264 consist of a domain-N, a putative transmembrane domain, and a possible cytoplasmic domain. The domain-N of W264 is 89% similar to that of NCA-50 at amino acid level, whereas the domain-N of W236 is only 49 and 43% similar to those of NCA-50 and pregnancy-specific beta 1-glycoprotein-11 (PSG11), respectively, indicating that W236 belongs to a new subfamily within the CEA family. The third clone W282 encodes a protein consisting of a domain-N virtually identical to that of W264 and a short hydrophilic C-terminal domain. W264 and W282 seem to be derived from a single gene by alternative splicing of RNA. These three new species are particularly unique in respect that they lack the repetitive immunoglobulin-related domains that have been universally found in the human CEA gene family members. The biochemical and immunochemical properties of the recombinant proteins of these cDNA clones, however, did not coincide with those of six NCA species previously identified in granulocytes at protein level, suggesting that, in granulocytes, there exist at least 12 NCA or NCA-related species whose expression is under complex control.     

Primary structure of rat liver dipeptidyl peptidase IV deduced from its cDNA and identification of the NH2-terminal signal sequence as the membrane-anchoring domain

Two forms of dipeptidyl peptidase IV (DPP) were purified from rat liver plasma membranes: a membrane form (mDPP) extracted with Triton X-100 and a soluble form (sDPP) prepared by treatment with papain. Apparent molecular masses of mDPP and sDPP were 109 and 105 kDa, respectively, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the two forms were found to be completely different from each other. For further information on the molecular structure, we constructed a lambda gt11 liver cDNA library and isolated two cDNA clones for DPP, lambda cDP37 and lambda cD5. The 3.5-kilobase cDNA insert of lambda cDP37 contains an open reading frame that encodes a 767-residue polypeptide with a calculated size of 88,107 Da, which is in reasonable agreement with that of DPP (87 kDa) immunoprecipitated from cell-free translation products. Eight potential N-linked glycosylation sites were found in the molecule, accounting for the difference in mass between the precursor and mature forms. Of particular interest is that the deduced NH2-terminal sequence with a characteristic signal peptide is completely identical to that determined for mDPP. In addition, the NH2-terminal sequence of sDPP is identified in the predicted sequence starting at the 35th position from the NH2 terminus. These results indicate that the signal peptide of DPP is not cleaved off during biosynthesis but functions as the membrane-anchoring domain even in the mature form. It is also found that the primary structure thus predicted has striking homology to that of gp 110, a bile canaliculus domain-specific membrane glycoprotein (Hong, W., and Doyle, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7962-7966).     

Structural analysis of the substrate recognition mechanism in O-phosphoserine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1

L-Cysteine is synthesized from O-acetyl-L-serine (OAS) and sulfide by O-acetylserine sulfhydrylase (OASS; EC 2.5.1.47) in plants and bacteria. O-phosphoserine sulfhydrylase (OPSS; EC 2.5.1.65) is a novel enzyme from the hyperthermophilic aerobic archaeon Aeropyrum pernix K1 (2003). OPSS can use OAS or O-phospho-L-serine (OPS) to synthesize L-cysteine. To elucidate the mechanism of the substrate specificity of OPSS, we analyzed three-dimensional structures of the active site of the enzyme. The active-site lysine (K127) of OPSS forms an internal Schiff base with pyridoxal 5'-phosphate. Therefore, crystals of the complexes formed by the K127A mutant with the external Schiff base of pyridoxal 5'-phosphate with either OPS or OAS were prepared and examined by X-ray diffraction analysis. In contrast to that observed for OASS, no significant difference was seen in the overall structure between the free and complexed forms of OPSS. The side chains of T152, S153, and Q224 interacted with the carboxylate of the substrates, as a previous study has suggested. The side chain of R297 has been proposed to recognize the phosphate group of OPS. Surprisingly, however, the position of R297 was significantly unchanged in the complex of the OPSS K127A mutant with the external Schiff base, allowing enough space for an interaction with OPS. The positively charged environment around the entrance of the active site including S153 and R297 is important for accepting negatively charged substrates such as OPS.     

Structure, function and evolution of the mitochondrial division apparatus

Mitochondria are derived from free-living alpha-proteobacteria that were engulfed by eukaryotic host cells through the process of endosymbiosis, and therefore have their own DNA which is organized using basic proteins to form organelle nuclei (nucleoids). Mitochondria divide and are split amongst the daughter cells during cell proliferation. Their division can be separated into two main events: division of the mitochondrial nuclei and division of the matrix (the so-called mitochondrial division, or mitochondriokinesis). In this review, we first focus on the cytogenetical relationships between mitochondrial nuclear division and mitochondriokinesis. Mitochondriokinesis occurs after mitochondrial nuclear division, similar to bacterial cytokinesis. We then describe the fine structure and dynamics of the mitochondrial division ring (MD ring) as a basic morphological background for mitochondriokinesis. Electron microscopy studies first identified a small electron-dense MD ring in the cytoplasm at the constriction sites of dividing mitochondria in the slime mold Physarum polycephalum, and then two large MD rings (with outer cytoplasmic and inner matrix sides) in the red alga Cyanidioschyzon merolae. Now MD rings have been found in all eukaryotes. In the third section, we describe the relationships between the MD ring and the FtsZ ring descended from ancestral bacteria. Other than the GTPase, FtsZ, mitochondria have lost most of the proteins required for bacterial cytokinesis as a consequence of endosymbiosis. The FtsZ protein forms an electron transparent ring (FtsZ or Z ring) in the matrix inside the inner MD ring. For the fourth section, we describe the dynamic association between the outer MD ring with a ring composed of the eukaryote-specific GTPase dynamin. Recent studies have revealed that eukaryote-specific GTPase dynamins form an electron transparent ring between the outer membrane and the MD ring. Thus, mitochondriokinesis is thought to be controlled by a mitochondrial division (MD) apparatus including a dynamic trio, namely the FtsZ, MD and dynamin rings, which consist of a chimera of rings from bacteria and eukaryotes in primitive organisms. Since the genes for the MD ring and dynamin rings are not found in the prokaryotic genome, the host genomes may make these rings to actively control mitochondrial division. In the fifth part, we focus on the dynamic changes in the formation and disassembly of the FtsZ, MD and dynamin rings. FtsZ rings are digested during a later period of mitochondrial division and then finally the MD and dynamin ring apparatuses pinched off the daughter mitochondria, supporting the idea that the host genomes are responsible for the ultimate control of mitochondrial division. We discuss the evolution, from the original vesicle division (VD) apparatuses to VD apparatuses including classical dynamin rings and MD apparatuses. It is likely that the MD apparatuses involving the dynamic trio evolved into the plastid division (PD) apparatus in Bikonta, while in Opisthokonta, the MD apparatus was simplified during evolution and may have branched into the mitochondrial fusion apparatus. Finally, we describe the possibility of intact isolation of large MD/PD apparatuses, the identification of all their proteins and their related genes using C. merolae genome information and TOF-MS analyses. These results will assist in elucidating the universal mechanism and evolution of MD, PD and VD apparatuses.     

Inhibition of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase by ß-D-4-O-sulfo-N-acetylgalactosaminides bearing various hydrophobic aglycons

N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO₄) residues of chondroitin sulfate to yield chondroitin sulfate E (CS-E). We have previously demonstrated that phenyl-ß-D-GalNAc(4SO₄) could serve as an acceptor for GalNAc4S-6ST, thereby inhibiting GalNAc4S-6ST competitively. In this paper we compared the inhibitory effects of various glycosides in which various hydrophobic aglycons were attached to D-GalNAc(4SO₄) via ß anomeric configuration. p-Nitrophenyl-ß-D-GalNAc(4SO₄) and p-chlorophenyl-ß-D-GalNAc(4SO₄) were stronger inhibitors than phenyl-ß-D-GalNAc(4SO₄). Among inhibitors examined here, 3-estradiol-ß-D-GalNAc(4SO₄) was the strongest inhibitor; the Ki of 3-estradiol-ß-D-GalNAc(4SO₄) for the competitive inhibition was 0.008 mM, which was much lower than the Ki of phenyl-ß-D-GalNAc(4SO₄), 0.98 mM. In contrast, 7-estradiol-ß-D-GalNAc(4SO₄) showed only weak inhibition to GalNAc4S-6ST. 3-Estradiol- ß-D-GalNAc(4SO₄) did not inhibit chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase under the concentration where GalNAc4S-6ST was inhibited by 90%. When 3-estradiol- ß-D-GalNAc(4SO₄) was added to the culture medium of chondrosarcoma cells expressing human GalNAc4S-6ST, a significant, albeit small, reduction in the cellular synthesis of CS-E was observed. These results suggest that estradiol group of 3-estradiol-ß-D-GalNAc(4SO₄) may enhance the inhibitory activity of the glycoside through increasing the affinity to the enzyme and may allow the glycosides to diffuse at a low efficiency into the cells to inhibit cellular synthesis of CS-E.