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Biomedical subjects

Masao Kawakita

Publications and source records attributed to Masao Kawakita.

At least 19 recordsLinked to original sources
PubMed2006

Role of proline residues in conferring thermostability on aqualysin I.

To understand the molecular basis of the thermostability of a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1, we introduced mutations at Pro5, Pro7, Pro240 and Pro268, which are located on the surface loops of aqualysin I, by changing these amino acid residues into those found at the corresponding locations in VPR, a psychrophilic serine protease from Vibrio sp. PA-44. All mutants were expressed stably and exhibited essentially the same specific activity as wild-type aqualysin I at 40 degrees C. The P240N mutant protein had similar thermostability to wild-type aqualysin I, but P5N and P268T showed lower thermostability, with a half-life at 90 degrees C of 15 and 30 min, respectively, as compared to 45 min for the wild-type enzyme. The thermostability of P7I was decreased even more markedly, and the mutant protein was rapidly inactivated at 80 degrees C and even at 70 degrees C, with half-lives of 10 and 60 min, respectively. Differential scanning calorimetry analysis showed that the transition temperatures of wild-type enzyme, P5N, P7I, P240N and P268T were 93.99 degrees C, 83.45 degrees C, 75.66 degrees C, 91.78 degrees C and 86.49 degrees C, respectively. These results underscore the importance of the proline residues in the N- and C-terminal regions of aqualysin I in maintaining the integrity of the overall protein structure at elevated temperatures.

Amino Acid Sequence↗
PubMed2006

Physical and functional association of human protein O-mannosyltransferases 1 and 2.

A defect of protein O-mannosylation causes congenital muscular dystrophy with brain malformation and structural eye abnormalities, so-called Walker-Warburg syndrome. Protein O-mannosylation is catalyzed by protein O-mannosyltransferase 1 (POMT1) and its homologue, POMT2. Coexpression of POMT1 and POMT2 is required to show O-mannosylation activity. Here we have shown that POMT1 forms a complex with POMT2 and the complex possesses protein O-mannosyltransferase activity. Results indicate that POMT1 and POMT2 associate physically and functionally in vivo. Recently, three mutations were reported in the POMT1 gene of patients who showed milder phenotypes than typical Walker-Warburg syndrome. We coexpressed these mutant POMT1s with POMT2 and found that none of them had any activity. However, all POMT1 mutants, including previously identified POMT1 mutants, coprecipitated with POMT2. These results indicate that the mutant POMT1s could form heterocomplexes with POMT2 but that such complexes are insufficient for enzymatic activity.

Abnormalities, Multiple↗
PubMed2006

Diacetylated derivatives of spermine and spermidine as novel promising tumor markers.

N1,N12-diacetylspermine (DiAcSpm) and N1,N8-diacetylspermidine (DiAcSpd) are minor components of human urinary polyamine to which little attention has been paid until recently. HPLC analysis of urinary polyamines has revealed that the excretion of these diacetylpolyamines, in particular, into urine was frequently and markedly increased in association with every type of cancer so far examined. Remission was usually accompanied by recovery of urinary diacetylpolyamines to the normal level. DiAcSpm was more sensitive than CEA for detecting colorectal cancer patients, while DiAcSpd was highly specific for malignant conditions in that the excretion of the latter was scarcely elevated in cases of benign urogenital diseases. An ELISA procedure for rapid determination of DiAcSpm was developed to promote the clinical application of these new tumor markers, and subsequent studies indicated that DiAcSpm was elevated in 60% of colorectal cancer patients at early stages (stage 0 + I), whereas only 10% of these patients were CEA-positive. DiAcSpm may also be useful as a follow-up marker that is efficient for detecting recurrence and sensitive to changes in the clinical condition of patients. The evidence accumulated so far indicates that DiAcSpm and DiAcSpd are promising novel tumor markers. They deserve more intensive studies, including studies of their biochemistry and metabolism.

Animals↗
PubMed2005

N(1),N(12)-Diacetylspermine as a sensitive and specific novel marker for early- and late-stage colorectal and breast cancers.

PURPOSE: N(1),N(12)-diacetylspermine (DiAcSpm) in the urine of colorectal and breast cancer patients was examined to establish its usefulness as a novel diagnostic tool for detecting these cancers at clinically early stages. EXPERIMENTAL DESIGN: Urine samples from 248 colon cancer patients and 83 breast cancer patients as well as 51 patients with benign gastrointestinal diseases treated in Tokyo Metropolitan Komagome Hospital during the period of August 1999 to January 2004 were collected. DiAcSpm was analyzed by ELISA and its sensitivity for malignant conditions was compared with that of serum carcinoembryonic antigen (CEA), CA19-9, and CA15-3. RESULTS: The sensitivity of urinary DiAcSpm for colon cancer patients (n = 248) was 75.8% (mean +/- 2 SD for 52 healthy controls as a cutoff value), which was markedly higher than the sensitivities of serum CEA (39.5%, P < 0.0001) and CA19-9 (14.1%, P < 0.0001). DiAcSpm was elevated in 60% of tumor-node-metastasis cancer stage 0 + I patients, whereas only 10% (P < 0.0001) and 5% (P < 0.0001) of these patients were CEA- and CA19-9-positive, respectively. The sensitivity of urinary DiAcSpm for 83 cases of breast cancer (60.2%) was higher than the sensitivities of CEA (37.3%, P = 0.0032) and CA15-3 (37.3%, P = 0.0032). DiAcSpm was elevated in 28% of tumor-node-metastasis stage I + II patients, whereas only 3% (P = 0.0064) and 0% (P = 0.001) of these patients were CEA- and CA15-3-positive, respectively. CONCLUSION: The observations indicate that urinary DiAcSpm is a more sensitive marker than CEA, CA19-9, and CA15-3 and that it can efficiently detect colorectal and breast cancers at early stages.

Adult↗
PubMed2005

Identification and characterization of human Golgi nucleotide sugar transporter SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family.

We report the molecular cloning of SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family. The gene SLC35D2 maps to chromosome 9q22.33. SLC35D2 cDNA codes for a hydrophobic protein consisting of 337 amino acid residues with 10 putative transmembrane helices. Northern blot analysis revealed the SLC35D2 mRNA as a single major band corresponding to 2.0 kb in length. SLC35D2 was localized in the Golgi membrane and exhibited around 50% similarity with three nucleotide sugar transporters: human SLC35D1 (UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter), fruitfly fringe connection (frc) transporter, and nematode SQV-7 transporter, the latter two being involved in developmental and organogenetic processes. Heterologous expression of SLC35D2 protein in yeast indicated that UDP-N-acetylglucosamine is a candidate for the substrate(s) of the transporter. The sequence similarity, subcellular localization, and transporting substrate suggest that SLC35D2 is a good candidate for the ortholog of frc transporter, which is involved in the Notch signaling system by providing the fringe N-acetylglucosaminyltransferase with the substrate. We also describe the identification and categorization of the human SLC35 gene family.

Amino Acid Sequence↗
PubMed2005

Interaction of N1,N12-diacetylspermine with polyamine transport systems of polarized porcine renal cell line LLC-PK1.

LLC-PK(1) cells grown on porous membrane filters were employed as a model system to explore the renal transport of polyamines. The polarity of LLC-PK(1) monolayers was confirmed by the exclusive appearance of a Na(+)-dependent alpha-methylglucoside transport system on the apical surface. The uptake of free polyamines from the basolateral side of monolayers was consistent with the existence of a single class of transport system, while the existence of two kinetically distinct polyamine transport systems with higher and lower affinities on apical membranes was suggested. The results of competition studies indicated that each of these transporters was able to interact with putrescine, spermidine and spermine. LLC-PK(1) cells incorporated monoacetylspermine from the apical surface of monolayers at about half the rate of spermine uptake. Monoacetylspermine inhibited spermidine uptake, indicating that free polyamine transport systems also recognized the monoacetylated derivative. In contrast, N(1),N(12)-diacetylspermine did not inhibit spermidine uptake, nor was it incorporated into the cells, indicating the absence of transport systems that recognize N(1),N(12)-diacetylspermine on the apical membranes of LLC-PK(1) cells. These results may be relevant as to our previous observation that the content of diacetylpolyamines in urine is relatively constant, and may explain the excellence of N(1),N(12)-diacetylspermine as a tumor marker.

Animals↗
PubMed2005

[Urinary diacetylspermine: its analysis and performance as a novel tumor marker].

N1,N12-Diacetylspermine (DiAcSpm) is excreted in the urine of healthy persons as a minor component of urinary polyamine. It is a promising tumor marker, since its excretion is frequently elevated in patients with various types of cancers. DiAcSpm was first detected and characterized by HPLC fractionation followed by enzymatic detection, but more recently, antibodies highly specific for DiAcSpm was prepared, and an ELISA system applicable to determination of urinary DiAcSpm was established. Measurement of urinary DiAcSpm using this ELISA system revealed that DiAcSpm is able to detect early stage (m and sm) colon cancers which CEA and CA19-9 cannot detect. DiAcSpm may also serve as a prognostic indicator and a marker for recurrence of colon cancer. Urinary DiAcSpm is elevated in metastatic and primary brain tumors including grade 3 and 4 gliomas and primary central nervous system lymphoma. In these primary brain tumors changes in urinary DiAcSpm were well correlated with the efficacy of treatments, recurrence of disease and increased malignancy of a tumor. DiAcSpm may be useful as a comprehensive indicator of the activeness of a brain tumor lesion in a patient. DiAcSpm was elevated in hepatocellular carcinoma, but patients with liver cirrhosis also showed considerably elevated levels of DiAcSpm.

Biomarkers, Tumor↗
PubMed2005

[Urine diacetylspermine as a novel tumor marker].

A urine tumor marker, diacetylspermine, was examined in patients with recurrent pancreato-biliary carcinoma, liver tumor, lung carcinoma and gynecologic malignancies. The urine marker increased together with recurrence, suggesting a recurrence monitoring marker at the outpatient ward. Regarding hepatocellular carcinoma, the sensitivity of the urine marker was as high as conventional markers such as AFP and PIVKA II. Synchronous examination of serum and urine markers showed a higher sensitivity than the single serum or urine marker for hepatocellular carcinoma. The sensitivity for non-advanced hepatocellular carcinoma was 50%, while that for advanced hepatocellular carcinoma was 83%. The urine marker may be useful to detect non-advanced hepatocellular carcinoma. The sensitivity for lung cancer was 83% and that for Stage I or II was 82%. Concerning uterine cervical tumor, the value of the urine marker increased with the grade of dysplasia. The sensitivity for ovarian carcinoma was 100%, while that for benign ovarian tumor was 0%. These findings suggest that urine diacetylspermine is a useful tumor marker in hepatocellular carcinoma, lung cancer and gynecologic malignancy as well as pancreatobiliary carcinoma.

Biliary Tract Neoplasms↗
PubMed2004

Reconstitution of GDP-mannose transport activity with purified Leishmania LPG2 protein in liposomes.

Activated nucleotide sugars required for the synthesis of glycoconjugates within the secretory pathway of eukaryotes are provided by the action of nucleotide sugar transporters (NSTs). Typically, NSTs are studied in microsomal preparations from wild-type or mutant lines; however, in this setting it can be difficult to assess NST properties because of the presence of glycosyltransferases and other interfering activities. Here we have engineered Leishmania donovani to express high levels of an active LPG2 Golgi GDP-Man transporter bearing a C-terminal polyhistidine tag. The functional LPG2-HIS was solubilized, purified by metal affinity chromatography, and reconstituted into phosphatidylcholine-containing liposomes using polystyrene SM-2 beads. The proteoliposomes exhibited robust GDP-Man transport activity with an apparent K(m) of 6.6 mum. Transport activity was enhanced by preloading of GMP and showed specificity for multiple substrates (GDP-Ara and GDP-Fuc). In contrast to the activity in crude microsomes, transport was not dependent on the presence of divalent cations. Thus, reconstitution of transport activity using purified LPG2 protein in liposomes provides firm experimental evidence that a single polypeptide is solely required for NST activity and is able to mediate the uptake of multiple substrates. These studies are relevant to the study of NST structure and function in both protozoan parasites as well as their higher eukaryotic hosts.

Animals↗
PubMed2004

Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates.

Cancer cells undergo distinct metabolic changes to cope with their hypoxic environment. These changes are achieved at least partly by the action of transcriptional factors called hypoxia-inducible factors (HIFs). We investigated gene expression in cultured human colon cancer cells induced by hypoxic conditions with special reference to cell-adhesion molecules and carbohydrate determinants having cell-adhesive activity by using DNA-microarray and RT-PCR techniques. Hypoxic culture of colon cancer cells induced a marked increase in expression of selectin ligands, the sialyl Lewis x and sialyl Lewis a determinants at the cell surface, which led to a definite increase in cancer cell adhesion to endothelial E-selectin. The transcription of genes for fucosyltransferase VII (FUT7), sialyltransferase ST3Gal-I (ST3O), and UDP-galactose transporter-1 (UGT1), which are all known to be involved in the synthesis of the carbohydrate ligands for E-selectin, was significantly induced in cancer cells by hypoxic culture. In addition, a remarkable induction was detected in the genes for syndecan-4 (SDC4) and alpha5-integrin (ITGA5), the cell-adhesion molecules involved in the enhanced adhesion of cancer cells to fibronectin. The transcriptional induction by hypoxia was reproduced in the luciferase-reporter assays for these genes, which were significantly suppressed by the co-transfection of a dominant-negative form of HIF. These results indicate that the metabolic shifts of cancer cells partly mediated by HIFs significantly enhance their adhesion to vascular endothelial cells, through both selectin- and integrin-mediated pathways, and suggest that this enhancement further facilitates hematogenous metastasis of cancers and tumor angiogenesis.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2004

[Clinical usefulness of urinary diacetylpolyamines as novel tumor markers].

N1,N12-Diacetylspermine(DiAcSpm) and N1,N8-diacetylspermidine(DiAcSpd) are excreted in the urine of healthy persons as minor components of urinary polyamine, with small individual variations in amount. They are promising tumor markers, since their excretion is frequently elevated in patients with various types of cancers. DiAcSpm is sensitive in cancer detection, while DiAcSpd is highly specific for cancer. Diacetylpolyamines were initially characterized and determined by HPLC fractionation, followed by enzymatic detection. More recently, antibodies highly specific for DiAcSpm and DiAcSpd were developed, and an ELISA system applicable to the determination of urinary DiAcSpm was established. Measurement of urinary DiAcSpm using this ELISA system revealed that DiAcSpm is a more sensitive tumor marker than CEA, CA19-9 and CA15-3 for colon and breast cancers. More importantly, DiAcSpm efficiently detects patients at early stages. On the other hand, CEA, CA19-9 and CA15-3 are quite insensitive for early stage cancers. The urinary DiAcSpm level tends to remain low even in tumor-bearing individuals when their cancerous lesions remain static, while it rises rapidly concomitant with recurrence. DiAcSpm may serve as a prognostic indicator and marker for recurrence of prostate and colon cancers. Diacetylpolyamines may turn out to be general tumor markers, since active proliferation of any cancer tissues would likely be accompanied by activation of polyamine metabolism.

Biomarkers, Tumor↗
PubMed2004

[The significance of urine di-acetyl spermine level as a cancer marker for colorectal cancer].

We analyzed the significance of the measurement of urine di-acetyl spermine (DiAcSpm) as a cancer marker for colorectal cancer treatment. We measured both the urine DiAcSpm(ELISA, normal range: 0-0.25 mumol/creatinine) and serum CEA (normal range: 0-5.0 ng/ml) of preoperative and postoperative colorectal cancer patients every month. We compared the positive rate from the cancer stage and the power of prognostic prediction. We divided the colorectal cancer patients into 4 groups: Group A: both levels were high; Group B: only the CEA level was high; Group C: only the DiAcSpm level was high; Group D: both levels were within a normal range. The positive rates of DiAcSpm and CEA from cancer staging were as follows: Stage 0: 62% and 9.5%, Stage I: 60% and 10%, Stage II: 70% and 42%, Stage III: 82% and 47%, and Stage IV: 88% and 63%, respectively. There was a significant difference (p < 0.0001) between both levels, especially for early-stage cases. The two-year survival rate was 0% in Group A, 100% in Group B, 72.7% in Group C and 100% in Group D. The difference among the 4 groups was significant (p < 0.0001). This showed that urine DiAcSpm predicted the prognosis after colorectal cancer surgery more exactly than serum CEA.

Biomarkers, Tumor↗
PubMed2004

[Urine diacetylspermine as a novel tumor marker for pancreatobiliary carcinomas].

A novel urine tumor marker, diacetylspermine, was compared with two conventional serum tumor markers, carcinoembryonic antigen (CEA) (highly specific for pancreatic cancer) and carbohydrate antigen (CA) 19-9 (highly sensitive for pancreatic cancer), in 125 patients with bilio-pancreatic tumors. When the diagnoses of benign or malignant conditions were examined, the sensitivity of urine diacetylspermine (75%) was shown to be higher than that of CEA (44%; P = 0.044) and CA19-9 (75%). The specificity of urine diacetylspermine (81%) was lower than that of CEA (92%) and as high as that of CA19-9 (80%). These results suggest that urine discetylspermine is a highly sensitive and specific novel marker for bilio-pancreatic carcinoma.

Adult↗
PubMed2003

Loss-of-function of an N-acetylglucosaminyltransferase, POMGnT1, in muscle-eye-brain disease.

Muscle-eye-brain disease (MEB), an autosomal recessive disorder, is characterized by congenital muscular dystrophy, brain malformation, and ocular abnormalities. Previously, we found that MEB is caused by mutations in the gene encoding the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), which is responsible for the formation of the GlcNAcbeta1-2Man linkage of O-mannosyl glycan. Although 13 mutations have been identified in patients with MEB, only the protein with the most frequently observed splicing site mutation has been studied. This protein was found to have no activity. Here, we expressed the remaining mutant POMGnT1s and found that none of them had any activity. These results clearly demonstrate that MEB is inherited as a loss-of-function of POMGnT1.

Abnormalities, Multiple↗
PubMed2003

Molecular physiology and pathology of the nucleotide sugar transporter family (SLC35).

The solute carrier family SLC35 consists of at least 17 molecular species in humans. The family members so far characterized encode nucleotide sugar transporters localizing at the Golgi apparatus and/or the endoplasmic reticulum (ER). These transporters transport nucleotide sugars pooled in the cytosol into the lumen of these organelles, where most glycoconjugate synthesis occurs. Pathological analyses and developmental studies of small, multicellular organisms deficient in nucleotide sugar transporters have shown these transporters to be involved in tumour metastasis, cellular immunity, organogenesis and morphogenesis. Leukocyte adhesion deficiency type II (LAD II) or the congenital disorder of glycosylation type IIc (CDG IIc) are the sole human congenital disorders known to date that are caused by a defect of GDP-fucose transport. Along with LAD II, the possible involvement of nucleotide sugar transporters in disorders of connective tissues and muscles is also discussed.

Animals↗
PubMed2003

Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum.

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.

Animals↗
PubMed2003

Substrate recognition by nucleotide sugar transporters: further characterization of substrate recognition regions by analyses of UDP-galactose/CMP-sialic acid transporter chimeras and biochemical analysis of the substrate specificity of parental and chimeric transporters.

Human UDP-Gal transporter 1 (hUGT1) and the human CMP-Sia transporter (hCST) are similar in structure, with amino acid sequences that are 43% identical, but they have quite distinct transport substrates. To define their substrate recognition regions, we constructed various chimeras between the two transporters and demonstrated that distinct submolecular regions of the transporter molecules are involved in the specific recognition of UDP-Gal and CMP-Sia (Aoki, K., Ishida, N., and Kawakita, M. (2001) J. Biol. Chem. 276, 21555-21561). In a further attempt to define the minimum submolecular regions required for the recognition of specific substrates, we found that substitution of helix 7 of hCST into the corresponding part of hUGT1 was necessary and sufficient for a chimera to show CST activity. Additional replacement of helix 2 or 3 of hUGT1 with the corresponding hCST sequence markedly increased the efficiency of CMP-Sia transport. For UGT activity, helices 1 and 8 of hUGT1 were necessary (but not sufficient), and helices 9 and 10 or helices 2, 3, and 7 derived from hUGT1 were also required to render the chimera competent for UDP-Gal transport. The in vitro analyses of a chimera with dual specificity indicated that it transported both UMP and CMP and mediated exchange reactions between these nucleotides and nucleotide sugars that are recognized specifically by either of the parental transporters.

Amino Acid Sequence↗