Biomedical subjects

M Fukuda

Publications and source records attributed to M Fukuda.

At least 361 records · Page 20Linked to original sources

PubMed1998

Synthesis of poly-N-acetyllactosamine in core 2 branched O-glycans. The requirement of novel beta-1,4-galactosyltransferase IV and beta-1,3-n-acetylglucosaminyltransferase.

Poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine repeats and provides the backbone structure for additional modifications such as sialyl Lex. Poly-N-acetyllactosamines in mucin-type O-glycans can be formed in core 2 branched oligosaccharides, which are synthesized by core 2 beta-1,6-N-acetylglucosaminyltransferase. Using a beta-1, 4-galactosyltransferase (beta4Gal-TI) present in milk and the recently cloned beta-1,3-N-acetylglucosaminyltransferase, the formation of poly-N-acetyllactosamine was found to be extremely inefficient starting from a core 2 branched oligosaccharide, GlcNAcbeta1-->6(Galbeta1-->3)GalNAcalpha-->R. Since the majority of synthesized oligosaccharides contained N-acetylglucosamine at the nonreducing ends, galactosylation was judged to be inefficient, prompting us to test novel members of the beta4Gal-T gene family for this synthesis. Using various synthetic acceptors and recombinant beta4Gal-Ts, beta4Gal-TIV was found to be most efficient in the addition of a single galactose residue to GlcNAcbeta1-->6(Galbeta1-->3)GalNAcalpha-->R. Moreover, beta4Gal-TIV, together with beta-1,3-N-acetylglucosaminyltransferase, was capable of synthesizing poly-N-acetyllactosamine in core 2 branched oligosaccharides. On the other hand, beta4Gal-TI was found to be most efficient for poly-N-acetyllactosamine synthesis in N-glycans. In contrast to beta4Gal-TI, the efficiency of beta4Gal-TIV decreased dramatically as the acceptors contained more N-acetyllactosamine repeats, consistent with the fact that core 2 branched O-glycans contain fewer and shorter poly-N-acetyllactosamines than N-glycans in many cells. These results, as a whole, indicate that beta4Gal-TIV is responsible for poly-N-acetyllactosamine synthesis in core 2 branched O-glycans.

Animals↗

PubMed1998

Fidelity levels of DNA polymerases in tumorigenic state cells and serially transplantable tumor cells.

It is well known that point mutations exist in oncogenes and tumor suppressor genes of tumor cells, and one of the causes of these mutations may be misincorporation by error-prone DNA polymerases. This hypothesis is supported by the observation of decreased fidelity levels of DNA polymerases in mouse spleen containing tumorigenic cells after infection with Friend virus, and in aged animals that suffer high rates of tumorigenesis. However, this decrease in fidelity is disadvantageous for tumor cells maintained by serial transplantation. Therefore, we measured the fidelity levels of DNA polymerases in tumor cells transplanted through many passages. The fidelity levels of DNA polymerases from Yoshida ascites hepatoma, Rhodamine sarcoma, mouse ascites hepatoma-134, and Ehrlich ascites carcinoma cells derived from rats and mice are very high for in-vitro DNA synthesis on synthetic polynucleotides. These results suggest that many kinds of mutant cells arise during tumorigenesis. Among these mutant cells, cells showing decreased DNA polymerase(s) fidelities are present and these cells may undergo cell death. On the other hand, cells with mutations in various oncogenes and tumor suppressor genes and without mutations in DNA polymerase genes may survive as serially transplantable tumor cells.

Animals↗

PubMed1998

Overexpression of branched O-linked oligosaccharides on T cell surface glycoproteins impairs humoral immune responses in transgenic mice.

The aberrant expression of core 2 O-glycans on T cell surface glycoproteins has been associated with various immunodeficient syndromes such as Wiskott-Aldrich syndrome and AIDS. To determine the effect of this aberrant expression of core 2 O-glycans on immune responses, we previously generated transgenic mice overexpressing core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) in T cells, and demonstrated that T cell primary immune responses mediated through interaction between T cells and antigen-presenting cells are impaired in the transgenic mice (Tsuboi, S., and Fukuda, M. (1997) EMBO J. 16, 6364-6373). In this study, we determined whether overexpression of core 2 oligosaccharides on T cells leads to impaired humoral immune responses by B cells using the same transgenic mice. When T cells were activated, both T and B cells from the transgenic and control mice expressed an equivalent amount of CD40L and CD40, which are, respectively, the receptor and counter-receptor for the interaction between T and B cells. However, activated T cells from the transgenic mice induced B cell proliferation less efficiently than those from control mice, regardless of whether B cells were isolated from control or the transgenic mice. This suggests that overexpression of core 2 O-glycans on T cell surface glycoproteins renders T cell-B cell interaction inefficient. Moreover, in the transgenic mice both immunoglobulin isotype switching and germinal center formation were also impaired. Taken together, these results indicate that aberrant expression of core 2 O-glycans on T cell surface glycoproteins results in impaired humoral immune responses due to an impaired interaction between T and B cells.

Animals↗

PubMed1998

Differential and cooperative polysialylation of the neural cell adhesion molecule by two polysialyltransferases, PST and STX.

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (NCAM), and these two polysialyltransferases often exist together in the same tissues. To determine the individual and combined roles of PST and STX in polysialic acid synthesis, in the present study we asked if PST and STX differ in the acceptor requirement and if PST and STX act together in polysialylation of NCAM. We first examined whether PST and STX differ in the requirement of sialic acid and core structures of N-glycans attached to NCAM. Polysialic acid was formed well on Lec4 and Lec13 cells, which are defective in N-acetylglucosaminyltransferase V and GDP-fucose synthesis, respectively, demonstrating that a side chain elongating from GlcNAcbeta1-->6Manalpha1-->6R and alpha-1,6-linked fucose are not required. PST and STX were found to add polysialic acid on NCAM.Fc molecules sialylated by alpha-2,3- or alpha-2,6-linkage in vitro, but not on NCAM.Fc lacking either sialic acid. These results indicate that both PST and STX have relatively broad specificity on N-glycan core structures in NCAM and no remarkable difference exists between PST and STX for the requirement of core structures and sialic acid attached to the N-glycans of NCAM. We then, using various N-glycosylation site mutants of NCAM, discovered that PST strongly prefer the sixth N-glycosylation site, which is the closest to the transmembrane domain, over the fifth site. STX slightly prefer the sixth N-glycosylation site over the fifth N-glycosylation site. The results also demonstrated that polysialic acid synthesized by PST is larger than that synthesized by STX in vitro. Moreover, a mixture of PST and STX more efficiently synthesized polysialic acid on NCAM than PST or STX alone. These results suggest that polysialylation of NCAM is influenced by the difference between PST and STX in their preference for N-glycosylation sites on NCAM. The results also suggest that PST and STX form polysialylated NCAM in a synergistic manner.

Animals↗

PubMed1998

The centrally acting beta1,6N-acetylglucosaminyltransferase (GlcNAc to gal). Functional expression, purification, and acceptor specificity of a human enzyme involved in midchain branching of linear poly-N-acetyllactosamines.

In the present experiments the cDNA coding for a truncated form of the beta1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67-74-kDa fusion protein. Typically a yield of 750 microg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc, converting the acceptor to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Accordingly, we call the recombinant beta1,6-GlcNAc transferase cIGnT6 to emphasize its action at central rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, beta1,4galactosyltransferase and beta1, 3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.

Amino Sugars↗

PubMed1998

A leukotriene receptor antagonist, ONO-1078, modulates drug sensitivity and leukotriene C4 efflux in lung cancer cells expressing multidrug resistance protein.

ONO-1078 is a new class of peptide leukotriene receptor antagonist, and multidrug resistance protein (MRP) is a membrane tranporter of multiple anticancer drugs and endogenous leukotriene C4 (LTC4). We investigated the effects of ONO-1078 on drug sensitivity and LTC4-efflux in MRP-expressing lung cancer cells. Drug sensitivity, intracellular vincristine accumulation, and intracellular and extracellular LTC4 concentrations were measured with or without ONO-1078. The effect of ONO-1078 on MRP-mediated calcein-efflux was determined by flow cytometry. ONO-1078 (1 to 10 microM) dose-dependently enhanced the sensitivity of NCI-H520 cells to vincristine with the reduced accumulation, and also enhanced the sensitivity to doxorubicin and etoposide. ONO-1078 inhibited both LTC4- and calcein-efflux from the cells with increased intracellular accumulations. Our findings indicate that ONO-1078 modulates multidrug resistance and inhibits LTC4-efflux in lung cancer cells, by inhibition of MRP function.

ATP-Binding Cassette Transporters↗

PubMed1998

Age dependent decline in the 3'-->5' exonuclease activity involved in proofreading during DNA synthesis.

A 3'-->5' exonuclease found in rat liver excises mispaired nucleotides at the 3'-hydroxyl end of primer chains such as poly dA-d(T9-C). Consequently, the priming activity of the chain from which the mispaired base was cut is greatly increased during DNA synthesis. These results suggest that the 3'-->5' exonuclease acts as a proofreading enzyme during DNA synthesis. The activity of this 3'-->5' exonuclease in the liver of 24-month-old rats is approximately 30% lower than the activity found in 4-month-old rats. Furthermore, non-complementary nucleotide incorporations by DNA polymerases from aged rats are observed during DNA synthesis on poly dA-dT10. The number of misincorporations decreases in the presence of the 3'-->5' exonuclease, but not all errors are prevented even when DNA polymerase and 3'-->5' exonuclease are added at an activity ratio similar to that found in vivo. The data suggest that declines in both the fidelity of DNA polymerase and the 3'-->5' exonuclease activity related to proofreading during the aging process lead to a higher frequency of base misincorporations during DNA replication.

Aging↗

PubMed1998

Efficient synthesis of 3'-glycosylated LacNAc-based oligosaccharides.

LacNAc-based oligosaccharides, including sialyl-(2-->3)-LacNAc, dimeric sialyl-(2-->3)-LacNAc, trimeric sialyl-(2-->3)-LacNAc, beta-glucuronyl-(1-->3)-LacNAc, and 3-sulfo-beta-glucuronyl-(1-->3)-LacNAc, were synthesized efficiently from a single protected LacNAc derivative having both OH-3' and 4' unprotected.

Amino Sugars↗

PubMed1998

Molecular cloning and expression of GDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism defective in Lec13 cells.

Subsets of mammalian cell surface oligosaccharides contain specific fucosylated moieties expressed in lineage- and/or temporal-specific patterns. The functional significance of these fucosylated structures is incompletely defined, although there is evidence that subsets of them, represented by the sialyl Lex determinant, are important participants in leukocyte adhesion and trafficking processes. Genetic deletion of these fucosylated structures in the mouse has been a powerful tool to address functional questions about fucosylated glycans. However, successful use of such approaches can be problematic, given the substantial redundancy in the mammalian alpha-1,3-fucosyltransferase and alpha-1,2-fucosyltransferase gene families. To circumvent this problem, we have chosen to clone the genetic locus encoding a mammalian GDP-D-mannose-4,6-dehydratase (GMD). This enzyme generates GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is then converted by the FX protein (GDP-4-keto-6-D-deoxymannose epimerase/GDP-4-keto-6-L-galactose reductase) to GDP-L-fucose. GMD is thus imperative for the synthesis of all fucosylated oligosaccharides. An expression cloning approach and the GMD-deficient CHO host cell line Lec13 were used to generate a population of cDNA molecules enriched in GMD cDNAs. This enriched plasmid population was then screened using a human expressed sequence tag (EST AA065072) with sequence similarity to an Arabidopsis thaliana GMD cDNA. This approach, together with 5'-rapid amplification of cDNA ends, yielded a human cDNA that complements the fucosylation defect in the Lec13 cell line. Northern blot analyses indicate that the GMD transcript is absent in Lec13 cells, confirming the genetic deficiency of this locus in these cells. By contrast, the transcript encoding the FX protein, which forms GDP-L-fucose from the ketosugar intermediate produced by GMD, is present in increased amounts in the Lec13 cells. These results suggest that metabolites generated in this pathway may participate in the transcriptional regulation of the FX protein and possibly the GMD protein. The results also suggest that the genomic structure encoding GMD in Lec13 cells likely has a defect different from a point mutation in the coding region.

Amino Acid Sequence↗

PubMed1998

Inositol 1,3,4,5-tetrakisphosphate binding activities of neuronal and non-neuronal synaptotagmins. Identification of conserved amino acid substitutions that abolish inositol 1,3,4,5-tetrakisphosphate binding to synaptotagmins III, V, and X.

Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10708-10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI-VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII-IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal synaptotagmins.

Amino Acid Sequence↗

PubMed1998

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.

Animals↗

PubMed1998

Nuclear export of actin: a novel mechanism regulating the subcellular localization of a major cytoskeletal protein.

Actin is a highly conserved, ubiquitous cytoskeletal protein, which is essential for multiple cellular functions. Despite its small size (Mr = 42 000), unpolymerized forms of actin, as well as polymerized forms, exist primarily in the cytoplasm, excluded from the nucleus. Although spatial control of actin is crucially important, the molecular mechanisms ensuring the cytoplasmic localization of unpolymerized actin have not been revealed so far. In this paper we report that actin contains two leucine-rich type nuclear export signal (NES) sequences in the middle part of the molecule, which are both shown to be functional. Monomeric actin, when injected into the nucleus, was rapidly exported in a manner which was sensitive to leptomycin B (LMB), a specific inhibitor of NES-dependent nuclear export. LMB treatment of cells prevented nuclear exclusion of endogenous actin, inducing its nuclear accumulation. Furthermore, actin mutants with disrupted NESs accumulated in the nucleus. Expression of these NES-disrupted actin mutants, but not of wild-type actin, induced a decrease in the proliferative potential of the cell. These results reveal a novel molecular mechanism controlling the subcellular distribution of actin.

3T3 Cells↗

PubMed1998

T-loop deletion of CDC2 from breast cancer tissues eliminates binding to cyclin B1 and cyclin-dependent kinase inhibitor p21.

The eukaryotic cell cycle is regulated by a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs). Monomeric free CDKs do not possess enzymatic activity, largely due to the steric hindrance caused by the T-loop at the entrance of the catalytic cleft, making ATP inaccessible to the substrate. Binding of a cyclin, primarily to the NH2-terminal lobe of the CDK that surrounds the PSTAIRE helix, induces a large conformational change in the PSTAIRE helix of the CDK and also causes the T-loop to move out of the way of the catalytic cleft. We identified from breast cancer tissues a novel variant of human CDC2, termed CDC2deltaT, that lacks 171 nucleotides corresponding to 57 amino acids, which compose most of the T-loop. CDC2deltaT was detected in 10 of 14 breast cancer tissues analyzed, whereas it was not detectable in diploid human fibroblast cell lines or in interleukin 2-stimulated normal human lymphocytes. CDC2deltaT protein is unable to complex with cyclin B1 and lacks histone H1 kinase activity. CDC2deltaT also fails to bind to the CDK inhibitor p21. These results indicate that the T-loop not only plays a key role in keeping a free CDK in its inactive state but also in facilitating CDK activation by promoting cyclin binding.

Alternative Splicing↗

PubMed1998

Interferon decreases hepatocellular carcinogenesis in patients with cirrhosis caused by the hepatitis B virus: a pilot study.

BACKGROUND: To elucidate the influence of long term interferon administration on the rate of occurrence of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV)-related cirrhosis, the authors analyzed 313 consecutive patients with cirrhosis. METHODS: Of the 313 patients, 94 underwent long term intermittent administration of interferon for > or = 6 months, and the remaining 219 patients received no interferon or other antiviral drug. RESULTS: Cumulative occurrence rates of HCC in the group treated with interferon and the untreated group were 4.5% and 13.3%, respectively, at the end of 3 years; 7.0% and 19.6%, respectively at the end of 5 years; and 17.0% and 30.8%, respectively, at the end of 10 years. The rate of HCC development in the treated group was significantly lower than that of the untreated group (P = 0.0124). The Cox proportional hazard model revealed that interferon treatment was an independent contributing factor in lowering the rate of carcinogenesis (odds ratio = 0.39; P = 0.031) even after correction by significant covariates in multivariate analysis. The virologic study showed that the role of interferon therapy from the viewpoint of cancer prevention was much more significant in patients with a HBV DNA concentration of > or = 10 milliequivalents. CONCLUSIONS: Interferon therapy for patients with HBV-related cirrhosis significantly decreased the HCC rate, especially in patients with a larger amount of serum HBV DNA. If interferon is administered properly for a selected group of patients, an effective strategy of cancer prevention can be achieved, even in patients with cirrhosis.

Adult↗

PubMed1998

Expression cloning of a human sulfotransferase that directs the synthesis of the HNK-1 glycan on the neural cell adhesion molecule and glycolipids.

The HNK-1 carbohydrate is expressed on various adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substratum interactions. Here we describe the isolation and functional expression of a cDNA encoding a human sulfotransferase that synthesizes the HNK-1 carbohydrate epitope. A mutant Chinese hamster ovary cell line, Lec2, which stably expresses human neural cell adhesion molecule (N-CAM) (Lec2-NCAM), was first established. Lec2-NCAM was co-transfected with a human fetal brain cDNA library, a cDNA encoding the rat glucuronyltransferase that forms a precursor of the HNK-1 carbohydrate, and a vector encoding the polyoma large T antigen. The transfected Lec2-NCAM cells expressing the HNK-1 glycan were enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA encoding a sulfotransferase, HNK-1ST, that directs the expression of the HNK-1 carbohydrate epitope on the cell surface. The deduced amino acid sequence indicates that the enzyme is a type II membrane protein. Sequence analysis revealed that there is a short amino acid sequence in the presumed catalytic domain, which is highly homologous to the corresponding sequence in other Golgi-associated sulfotransferases so far cloned. The amount of HNK-1ST transcript is high in fetal brain compared with fetal lung, kidney, and liver. Expression of HNK-1ST resulted in the formation of the HNK-1 epitope on N-CAM and a soluble chimeric form of HNK-1ST was shown to add a sulfate group to a precursor, GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, forming sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R. The results combined together indicate that the cloned HNK-1ST directs the synthesis of the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the nervous tissues.

Amino Acid Sequence↗

PubMed1998

Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300.

It has been hypothesized that certain viral infections directly activate a transcription factor(s) which is responsible for the activation of genes encoding type I interferons (IFNs) and interferon-stimulated genes (ISGs) via interferon regulatory factor (IRF) motifs present in their respective promoters. These events trigger the activation of defense machinery against viruses. Here we demonstrate that IRF-3 transmits a virus-induced signal from the cytoplasm to the nucleus. In unstimulated cells, IRF-3 is present in its inactive form, restricted to the cytoplasm due to a continuous nuclear export mediated by nuclear export signal, and it exhibits few DNA-binding properties. Virus infection but not IFN treatment induces phosphorylation of IRF-3 on specific serine residues, thereby allowing it to complex with the co-activator CBP/p300 with simultaneous nuclear translocation and its specific DNA binding. We also show that a dominant-negative mutant of IRF-3 could inhibit virus-induced activation of chromosomal type I IFN genes and ISGs. These findings suggest that IRF-3 plays an important role in the virus-inducible primary activation of type I IFN and IFN-responsive genes.

Amino Acid Sequence↗

PubMed1998

Effect of a 3'-->5' exonuclease with a proofreading function on the fidelity of error-prone DNA polymerase alpha from regenerating liver of aged rats.

A nuclease that releases noncomplementary nucleotides from the 3'-end of DNA was isolated and highly purified from rat liver extract. The d(T9-C) priming activities for DNA synthesis in vitro by DNA polymerases alpha and beta were recovered by the addition of this enzyme, which itself does not contain a DNA polymerase activity. This nuclease hydrolysed nucleotides from the 3'-end, but did not remove [32P]-labeled nucleotides from the 5'-terminus of specifically labeled DNA. Also, the reaction products released from the 3'-end of DNA were all mononucleotides. These results indicate that the exonuclease is a 3'-->5' exonuclease with properties the same as those of DNase VII from human placenta. Rat DNase VII requires 4 mM MgCl2 or 0.125 mM MnCl2 for maximum activity, and shows a pH optimum of 7.5. These optimal conditions are similar to those of DNA polymerases, and indicate that both rat DNase VII and DNA polymerases are able to act under same conditions. Non-complementary nucleotide incorporation by DNA polymerase alpha from aged rat has been observed during in vitro DNA synthesis on poly dA-dT10. The amount of this mis-incorporation is decreased by the coexistence of the 3'-->5' exonuclease, but not all errors are edited out. Thus, this rat DNase VII is suggested to play an important role in proofreading during DNA synthesis.

Aging↗

PubMed1998

Molecular cloning and expression of a novel human trans-Golgi network glycoprotein, TGN51, that contains multiple tyrosine-containing motifs.

Previously, it has been shown that glycoproteins with approximately 130-kDa molecular mass react with antisera from patients with renal vasculitis (Kain, R., Matsui, K., Exner, M., Binder, S., Schaffner, G., Sommer, E. M., and Kerjaschki, D. (1995) J. Exp. Med. 181, 585-597). To search for a molecule that reacts with the antibodies, we screened a lambdagt11 human placental cDNA library. Two of the isolated clones were found to encode a putative counterpart of the rodent trans-Golgi network (TGN) glycoprotein 38, hTGN46, which has the tyrosine containing motif YQRL shared by mouse and rat TGN38. Moreover, reverse transcription-polymerase chain reaction analysis of hTGN46 transcripts and genomic analysis of a cDNA deposited as an expressed sequence tag in dbEST Data Base revealed that additional cDNAs exist that are produced by alternate usage of 3'-splice sites of intron III. Alternative splicing results in frame shifts and leads to novel larger translation products with one (for hTGN48) or two (for hTGN51) additional tyrosine-containing motifs. hTGN51 expressed in Chinese hamster ovary cells were localized to the trans-Golgi network, overlapping with beta-1,4-galactosyltransferase even after mutating the tyrosine-containing motif common to hTGN46. In contrast, mutated hTGN48 and hTGN46 are no longer retrieved to the TGN. These results strongly suggest that hTGN51 may have a unique function compared with hTGN46 or hTGN48 in shuttling between the cell surface and the TGN.

Amino Acid Sequence↗