A novel apolipoprotein C-II mimetic peptide that activates lipoprotein lipase and decreases serum triglycerides in apolipoprotein E-knockout mice.

Apolipoprotein A-I (apoA-I) mimetic peptides are currently being developed as possible new agents for the treatment of cardiovascular disease based on their ability to promote cholesterol efflux and their other beneficial antiatherogenic properties. Many of these peptides, however, have been reported to cause transient hypertriglyceridemia due to inhibition of lipolysis by lipoprotein lipase (LPL). We describe a novel bihelical amphipathic peptide (C-II-a) that contains an amphipathic helix (18A) for binding to lipoproteins and stimulating cholesterol efflux as well as a motif based on the last helix of apolipoprotein C-II (apoC-II) that activates lipolysis by LPL. The C-II-a peptide promoted cholesterol efflux from ATP-binding cassette transporter ABCA1-transfected BHK cells similar to apoA-I mimetic peptides. Furthermore, it was shown in vitro to be comparable to the full-length apoC-II protein in activating lipolysis by LPL. When added to serum from a patient with apoC-II deficiency, it restored normal levels of LPL-induced lipolysis and also enhanced lipolysis in serum from patients with type IV and V hypertriglyceridemia. Intravenous injection of C-II-a (30 mg/kg) in apolipoprotein E-knockout mice resulted in a significant reduction of plasma cholesterol and triglycerides of 38 ± 6% and 85 ± 7%, respectively, at 4 hours. When coinjected with the 5A peptide (60 mg/kg), the C-II-a (30 mg/kg) peptide was found to completely block the hypertriglyceridemic effect of the 5A peptide in C57Bl/6 mice. In summary, C-II-a is a novel peptide based on apoC-II, which promotes cholesterol efflux and lipolysis and may therefore be useful for the treatment of apoC-II deficiency and other forms of hypertriglyceridemia.

[Case of spuriously high level of squamous cell carcinoma antigen in patient without carcinoma--HPLC and ultrafiltration are useful to analyze the true value].

Squamous cell carcinoma antigen (SCCA) is a diagnostic tumor marker for the advanced uterine, cervix and lung tumor. Although SCCA is a prognostic indicator for some tumors, recent progress of this marker has shown that the SCCA could also be found in the serum of nonmalignant disease such as renal failure and others. Here, we report a case of spuriously high level of SCCA in patient without carcinoma, renal failure, head-and-neck disease and lung disease. An early fifties female who had been undergone the diagnostic conization for high-grade cervical intraepithelial neoplasia ten years ago and observed without special treatment with around 20ng/ml level of SCCA. She has no signs of tumor, renal failure, head-and-neck disease or lung disease until now. The high performance liquid chromatography with Superdex 200 showed the molecular weight of the major part of SCCA of the patient is more than 160 kDa and the part of 45 kDa, the same molecular weight as lung tumor, is trace amount. Moreover, the ultrafiltration analysis showed the SCCA of the present case did not penetrate the 100 kDa cut-filter, but SCCAs with other patients with uterine, cervix, lung tumor and renal failure did penetrate the filter. In this case, the analysis of molecular weight of SCCA using HPLC gel filtration and ultrafiltration is useful to rule out spuriously elevated SCCA.

Composition of plasmalogens in serum lipoproteins from patients with non-alcoholic steatohepatitis and their susceptibility to oxidation.

BACKGROUND: Plasmalogens are ether phospholipids (PL) with an alkenyl group including vinyl ether bound at the sn-1 position and a polyunsaturated fatty acid bound at the sn-2 position, and are susceptible to oxidation. To date, there are no reports on the relationship between plasmalogen in serum lipoproteins and non-alcoholic steatohepatitis (NASH), caused by multiple factors including oxidative stress. Here, we have investigated the distribution of plasmalogens in serum lipoproteins isolated from NASH patients and healthy volunteers. METHODS: Serum lipoproteins were separated by gel-filtration chromatography, and analyzed for ethanolamine and choline plasmalogens using liquid chromatography-mass spectrometry. RESULTS: Both plasmalogen levels were higher in HDL than in VLDL or LDL. The plasmalogens/PL ratio was significantly lower in NASH than controls, for all lipoprotein fractions. Ethanolamine plasmalogens containing 20:4 and 22:6 at the sn-2 position and choline plasmalogens containing 16:0 at the sn-1 position were predominant in each group. In oxidation test using LDL from healthy serum, both types of plasmalogens were decreased during the early stages of oxidation. CONCLUSION: Plasmalogens could be a potential biomarker for evaluating the early stages of oxidation in NASH.

Primary relapse of acute lymphoblastic leukemia in a cervical smear: a case report.

Uterine cervix and corpus are rarely the initial site of relapse in leukemia or lymphoma. We report herein a case of uterine cervical relapse with B-cell acute lymphoblastic leukemia (ALL). The patient, a 60-yr-old woman, had a history of ALL that had been in remission for 2 yr after chemotherapy. She presented with a chief complaint of genital bleeding. In a routine cervico-vaginal Papanicolau smear, abundant atypical lymphoid cells with round-to-oval nuclei, scant cytoplasm, and high nuclear to cytoplasmic ratios was observed. The nuclei of these cells had fine and dark chromatin and thickened nuclear membranes, with one or several nucleoli being visible. Biopsy under colposcope was performed, and a diagnosis of relapse of ALL was confirmed. The ongoing genital bleeding presented a problem with clinical management of the patient. It was decided to proceed with hysterectomy to end that problem and thereafter proceed with therapy directed against the leukemia. Our results suggest that in patients with known extrauterine cancer, the presence of malignancy in uterine cellular samples provides information regarding the extent of the neoplasm.

Management of transvaginal ultrasound-guided absolute ethanol sclerotherapy for ovarian endometriotic cysts.

PURPOSE: Endometriosis and endometriotic ovarian cysts are common gynecologic diseases. Excision of the cyst wall by laparotomy or laparoscopy is the standard treatment for endometriotic ovarian cysts. However, some young patients with cysts would prefer not to have an abdominal incision. We reviewed and assessed the effectiveness of transvaginal ultrasound-guided ethanol sclerotherapy in these patients. METHODS: Eighteen patients with endometriotic ovarian cysts underwent transvaginal ultrasound-guided aspiration and ethanol sclerotherapy using spinal anesthesia. The contents of the cysts were drained and sent for cytological examination. The cyst cavities were washed with absolute ethanol, and the cysts were then filled with absolute ethanol for 5 min. The patients were followed up with transvaginal ultrasonography. RESULTS: The transvaginal ethanol sclerotherapy was completed in all cases. The mean long diameter of the cysts was 50 mm (31-100 mm), and the mean operative duration was 22 min (8-45 min). Malignant cells were not isolated from the aspirated fluid in any case. There were no significant intra- or postoperative complications. Two patients (11.1%) had a recurrence at 3 and 32 months after ethanol sclerotherapy, respectively. CONCLUSIONS: Transvaginal ultrasound-guided absolute ethanol sclerotherapy is useful for ovarian endometriotic cysts, particularly in young patients or in patients who would like to become pregnant. However, careful selection based on ultrasonography or magnetic resonance imaging findings and the age of the patients is critical.

Benign endometrial adenofibroma and polyp in patients receiving tamoxifen: findings on transvaginal ultrasonography and magnetic resonance imaging.

Tamoxifen, a selective estrogen receptor modulator, is widely used to treat breast cancer, but an association has been reported between tamoxifen and the development of endometrial lesions, including endometrial carcinoma, endometrial polyps, and endometrial hyperplasia. There have also recently been a few reports on the relation between tamoxifen and adenofibroma. We present two case reports, one of a patient with a uterine adenofibroma and one of a patient with an endometrial polyp, both of whom received tamoxifen. Cases 1 and 2 are 75- and 65-year-old postmenopausal women, respectively, undergoing tamoxifen therapy. In both cases, endometrial thickening and many small cysts in the uterine cavity were revealed by transvaginal ultrasonography or magnetic resonance imaging. Postoperative microscopic examination confirmed the mass as an adenofibroma in case 1 and as an endometrial polyp in case 2.

Control of mechanical properties of chitin nanofiber film using glycerol without losing its characteristics.

Surface-deacetylated chitin nanofiber films plasticized with glycerol were prepared to control mechanical properties. Nanofiber networks were able to retain excessive glycerol content up to 70% to obtain self-standing film. All films were flexible and highly transparent independent of glycerol content. Glycerol significantly decreased the Young's moduli and tensile strengths, and increased the fracture strain due to its plasticizing effect. At the same time, glycerol did not change the high transparency or the low thermal expansion of the nanofiber film.

Serum choline plasmalogens-those with oleic acid in sn-2-are biomarkers for coronary artery disease.

BACKGROUND: Identifying risk factors is crucial for preventing cardiovascular events, but there are no widely accepted predictive biomarkers. In our previous study of Japanese asymptomatic cohorts, we performed global analysis of serum ether glycerophospholipids (Egp) molecular profiles, and found that choline plasmalogens (PlsCho; 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphocholine), particularly those containing oleic acid (18:1) in the sn-2 position, were strongly associated with a wide range of risk factors for metabolic syndrome/atherosclerosis. METHODS: We determined serum concentrations of Egp molecular species of coronary artery disease patients (n=50; 31 males and 19 females) by LC/MS/MS, and plasmalogen (Pls; 1-O-alk-1'-enyl-2-acyl-sn-glycerophospholipids) contents in lipoprotein fractions by HPLC using radioactive iodine. RESULTS: We found that the serum concentrations of ether choline glycerophospholipids (EgpCho), particularly PlsCho, were not only significantly lower in males with significant coronary stenosis but also associated with atherosclerosis-related parameters, and their association was stronger than either high-density lipoprotein cholesterol or adiponectin. In addition, serum PlsCho containing 18:1 or linoleic acid (18:2) in sn-2 showed the highest correlations with a wide range of atherogenic parameters among PlsCho molecular species. CONCLUSION: These results verify our previous findings that serum PlsCho, particularly those containing 18:1 in sn-2, may serve as reliable biomarkers for atherosclerosis.

Preparation of high-strength transparent chitosan film reinforced with surface-deacetylated chitin nanofibers.

Surface-deacetylated chitin nanofiber reinforced chitosan films were prepared. The nano-composite films were highly transparent of approximately 84% at 600 nm due to the nanometer-sized fillers and chitosan matrix, which were embedded in the cavities and on the rough surface of the nanofiber networks. Due to the extended crystalline structure, the nanofibers worked effectively as reinforcement filler to improve the Young's modulus and the tensile strength of the chitosan film. After 10% blending of nanofiber, these properties were increased by 65% and 94%, respectively. Moreover, thermal expansion was also significantly decreased from 35.3 to 26.1 ppm K(-1) after 10% addition of nanofibers. Surface-deacetylated chitin nanofiber and the nano-composite films showed antifungal activity against A. alternata.

Immunological detection of large oxidized lipoproteins in hypertriglyceridemic serum.

BACKGROUND: Triglyceride-rich, low-density lipoproteins (TG-rich LDL) have been reported as an oxidized lipoprotein species in patients with severe liver disease. Using TG-rich LDL as an immunogen, we obtained a monoclonal antibody (G11-6) that reacted with TG-rich LDL from patients with liver disease and with metal-oxidized LDL only in the early process of the oxidation reaction. This study determined the G11-6-reactive lipoproteins in hypertriglyceridemic serum. METHODS: Serum samples from healthy volunteers (n = 12) and hypertriglyceridemic patients (n = 9) were fractionated by gel filtration and subjected to a sandwich enzyme-linked immunosorbent assay (ELISA) using G11-6 and polyclonal anti-apolipoprotein B antibodies. RESULTS: Small LDL and larger lipoproteins reacted with G11-6. G11-6-reactive small LDL was identified in both the healthy subjects and hypertriglyceridemic patients, whereas G11-6-reactive larger lipoproteins were found only in the hypertriglyceridemic patients. CONCLUSIONS: G11-6 is a useful tool for detecting increased large oxidized lipoproteins in hypertriglyceridemic patients.

Measurement of single low-density lipoprotein particles by atomic force microscopy.

BACKGROUND: The size of lipoprotein particles is relevant to the risk of coronary artery disease (CAD). METHODS: We investigated the feasibility of atomic force microscopy (AFM) for evaluating the size of large low-density lipoprotein (LDL) and small dense LDL (sd-LDL) separated by ultracentrifugation. The measurements by AFM in tapping mode were compared to those by electron microscopy (EM). RESULTS: There was a significant difference in particle sizes determined by AFM between large LDL (20.6 ± 1.9 nm, mean ± SD) and sd-LDL (16.2 ± 1.4 nm) obtained from six healthy volunteers (P < 0.05). The particle sizes determined by EM for the same samples were 23.2 ± 1.4 nm for large LDL and 20.4 ± 1.4 nm for sd-LDL. The difference between large LDL and sd-LDL detected by EM was also statistically significant (P < 0.05). In addition, the particle sizes of each lipoprotein fraction were significantly different between AFM and EM: P < 0.05 for large LDL and P < 0.05 for sd-LDL. CONCLUSIONS: AFM can differentiate between sd-LDL and large LDL particles by their size, and might be useful for evaluating risk for CAD.

Preparation of polysilsesquioxane-urethaneacrylate copolymer film reinforced with chitin nanofibers.

Chitin nanofibers (CNFs) reinforced silsesquioxane-urethaneacrylate (SSQ-UA) copolymer films were prepared. CNFs-SSQ-UA nanocomposite films were highly transparent due to the filling of nanometer sized (10-20 nm) CNFs inside the hybrid organic-inorganic SSQ-UA copolymer. CNFs due to their crystalline structure drastically increased Young's moduli and the tensile strengths of the composite and decreased the thermal expansion. High thermal stability of polysilsesquioxane improved heat resistance of CNFs.

Novel monoclonal antibody recognizing triglyceride-rich oxidized LDLs associated with severe liver disease and small oxidized LDLs in normal subjects.

BACKGROUND: Triglyceride-rich low-density lipoproteins (TG-rich LDLs) in the plasma of patients with severe liver disease are reported to change macrophages into foam cells in vitro. METHODS: Male BALB/c mice were immunized with TG-rich LDLs isolated from the plasma of a patient with severe liver disease. The resulting monoclonal antibody (G11-6) was used in a sandwich enzyme-linked immunosorbent assay (ELISA) in combination with polyclonal anti-apolipoprotein B antibodies. The time course of copper-mediated LDL oxidation was monitored using this ELISA. The results were compared with those of the two commercial ELISAs for oxidized LDLs using DLH or ML25, thiobarbituric acid reactive substances and the optical absorbance for the conjugated dienes generated in lipid peroxides. Furthermore, the lipoprotein fractions separated by gel filtration were tested with this ELISA in healthy volunteers (n = 11) and patients (n = 3) with liver disease. RESULTS: G11-6 reacted with oxidized LDLs during only the early phase of copper oxidation, being distinct from the other monoclonal antibodies and methods. G11-6 was confirmed to react with TG-rich LDLs in patients, while it reacted with small LDL particles in normal controls. CONCLUSIONS: The monoclonal antibody G11-6 is useful for detecting oxidized small LDLs in normal controls and oxidized TG-rich LDLs in patients with severe liver disease.

Endometrial clear cell adenocarcinoma diagnosed by endometrial cytological examination: two cases report.

In contrast to endometrioid endometrial adenocarcinoma (EA), endometrial clear cell adenocarcinoma (ECCA) is rare and shows a clinically aggressive potential, resulting in a poor prognosis. Therefore, the early diagnosis of ECCA is crucial for the treatment of patients bearing the tumor. Here, we report two cases of ECCA diagnosed by cytological examination. Case 1 was a 67-year-old female, while case 2 was a 68-year-old female. In both cases, tumors of the corpus uteri were identified, and they were diagnosed as ECCA on cytological examination with specimens derived from endometrial brushing. The diagnosis was confirmed by the histological examination of the surgical specimens.

Vaginal Lymphoma with Immune Thrombocytopenic Purpura: An Unusual Case Report.

The female genital tract is rarely the initial site of presentation in lymphoma or leukemia. We report a case of non-Hodgkin's lymphoma (NHL) presenting initially in the vagina. The patient, a 75-year-old woman, had a history of immune thrombocytopenic purpura (ITP). She presented with a chief complaint of genital bleeding and introital pain. On transvaginal ultrasonography, a vaginal tumor with an irregular wall was detected, and the internal echo showed a hypoechoic and echogenic pattern. Ultrasonography and magnetic resonance imaging (MRI) suggested that the vaginal tumor was likely to be a hematoma or a hemorrhagic tumor arising from ITP. Incision and resection for a hematoma or a hemorrhagic tumor were carried out in response to genital bleeding, introital pain, and pathological diagnosis. Postoperative microscopic examination confirmed that the tumor was a vaginal NHL. The final diagnosis using the Ann Arbor staging system was high-stage (stage IV) NHL. The patient received chemotherapy, and she remains in remission for 42 months after treatment.

Regulation of Notch signaling by dynamic changes in the precision of S3 cleavage of Notch-1.

Intramembrane proteolysis by presenilin-dependent gamma-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-beta. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val-->Gly and the Lys-->Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

Presenilin-dependent gamma-secretase on plasma membrane and endosomes is functionally distinct.

The presenilin (PS)/gamma-secretase complex, which contains not only PS but also Aph-1, PEN-2, and nicastrin, mediates proteolysis of the transmembrane domain of beta-amyloid protein precursor (betaAPP). Intramembrane proteolysis occurs at the interface between the membrane and cytosol (epsilon-site) and near the middle of the transmembrane domain (gamma-site), generating the betaAPP intracellular domain (AICD) and Alzheimer disease-associated Abeta, respectively. Both cleavage sites exhibit some diversity. Changes in the precision of gamma-cleavage, which potentially results in secretion of pathogenic Abeta42, have been intensively studied, while those of epsilon-cleavage have not. Although a number of PS-associated factors have been identified, it is unclear whether any of them physiologically regulate the precision of cleavage by PS/gamma-secretase. Moreover, there is currently no clear evidence of whether PS/gamma-secretase function differs according to the subcellular site. Here, we show that endocytosis affects the precision of PS-dependent epsilon-cleavage in cell culture. Relative production of longer AICDepsilon49 increases on the plasma membrane, whereas that of shorter AICDepsilon51 increases on endosomes; however, this occurs without a concomitant major change in the precision of cleavage at gamma-sites. Moreover, very similar changes in the precision of epsilon-cleavage are induced by alteration of the pH. Our findings demonstrate that the precision of epsilon-cleavage by PS/gamma-secretase changes depending upon the conditions and the subcellular location. These results suggest that the precision of cleavage by the PS/gamma-secretase complex may be physiologically regulated by the subcellular location and conditions.

Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain.

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.

Expression of the endothelial cell differentiation gene 7 (EDG-7), a lysophosphatidic acid receptor, in ovarian tumor.

AIM: Lysophosphatidic acid (LPA) has received attention as a mitogen because the physiologically active lipid stimulates ovarian cancer cell growth by interacting with specific receptors, the endothelial cell differentiation gene (EDG) family. In the present study, we have investigated the expression of EDG-7 mRNA, part of the EDG family, in both human ovarian cancers and established human ovarian cancer cell lines. METHODS: RNA was extracted from six ovarian cancer cell lines and multiple cancerous and normal ovarian tissues. The expression of EDG-7 mRNA was measured using reverse transcription-polymerase chain reaction and northern blotting, using reduced glyceraldehyde-phosphate dehydrogenase and S26 as internal controls. RESULTS: Of the cell lines tested, EDG-7 mRNA was expressed most intensely in CRL-11731 and CRL-1572 and at a lesser but still substantial level in CRL-11732. The expression of EDG-7 mRNA was limited in MCAS, CRL-11730 and TYKnu. In the ovarian cancer tissues, EDG-7 mRNA was expressed most highly in endometrioid adenocarcinoma and serous cystadenocarcinoma. The expression of EDG-7 mRNA was limited in clear cell adenocarcinoma and undetectable in mucinous cystadenocarcinoma. CONCLUSIONS: The intense EDG-7 expression in ovarian cancers suggests that the relation between LPA and EDG-7 (an LPA receptor) is involved in cancer cell growth and proliferation in some histologic subtypes of ovarian cancer.

Correlation p53 expression and human papilloma virus deoxyribonucleic acid with clinical outcome in early uterine cervical carcinoma.

BACKGROUND: In the present study we assessed whether expression of p53 protein or HPV DNA correlates with recurrence as well as several known prognostic factors in uterine cervical carcinoma. METHODS: Forty-nine patients with FIGO stage IA-IIB who underwent hysterectomy between 1998 and 2002 were retrospectively studied. All 49 cancer tissue samples were used for immunohistochemical study. Twenty-five of 49 cases were also examined by PCR-RFLP for detection and typing of HPV DNA. RESULTS: Twenty of 49 (40.8%) specimens demonstrated nuclear staining for p53. A significant association between p53 overexpression and age, hormonal status, FIGO stage, or recurrence was observed (p=0.02, 0.01, 0.03, 0.01). However, no significant association was found between p53 overexpression and lymph node metastases, parametrium involvement, or risk of death (p=0.18, 0.06, 0.14). Nineteen of 25 (76%) were HPV DNA-positive and 6 (24%) were negative. DISCUSSION: There was no relation between HPV DNA positivity and age, FIGO stage, lymph node metastases, parametrium involvement, recurrence, or risk of death. CONCLUSION: p53 overexpression is associated with age, hormonal status, FIGO stage, and recurrence in uterine cervical carcinoma.