Brain-derived neurotrophic factor induces angiogenin secretion and nuclear translocation in human umbilical vein endothelial cells.

Brain-derived neurotrophic factor (BDNF) is a well-known humoral protein that induces growth of neurons. Recent studies have suggested that BDNF could act as an angiogenesis inducer similar to vascular endothelial growth factor (VEGF). Angiogenin is a strong mediator of angiogenesis. It has particular characteristics both as a secreted protein and a transcription factor. After being incorporated into the cytoplasm, angiogenin is immediately transferred to the nucleus and then mediates the angiogenic effects of angiogenesis inducers, including VEGF. The aim of this study is to determine the association between BDNF and angiogenin. At first, we determined the secretion of angiogenin from human umbilical vein endothelial cells (HUVEC) induced by BDNF with enzyme-linked immunosorbent assay. Next, we determined BDNF-induced nuclear translocation of angiogenin by immunofluorescent staining. In addition, we examined the mRNA expression of angiogenin in HUVEC before and after BDNF stimulation by quantitative reverse transcriptase-polymerase chain reaction. As a result, we noted that BDNF induced angiogenin secretion and nuclear translocation without an increase in the mRNA expression in HUVEC. Furthermore, we demonstrated that BDNF-induced HUVEC proliferation was significantly suppressed when neomycin, a specific inhibitor of nuclear translocation of angiogenin, was administered. These findings indicate that nuclear translocation of angiogenin is critically involved in BDNF-induced proliferation of HUVEC. In conclusion, angiogenin contributes to angiogenesis induced by BDNF.

Transient human anti-mouse antibody generated with immune enhancement in a carbohydrate antigen 19-9 immunoassay after surgical resection of recurrent cancer.

UNLABELLED: We report a case of transient human anti-mouse antibody from a 64-year-old man in a carbohydrate antigen 19-9 immunoassay using an AIA 1800 analyser that generated immune enhancement after surgical resection of recurrent cancer. METHODS: The carbohydrate antigen 19-9 concentration was measured using an AIA 1800 analyser and a UniCel Dxl 800. Size-exclusion high-performance liquid chromatography was carried out on a Superose 12 column to estimate the carbohydrate antigen 19-9 elution profile using an AIA 1800 analyser. To determine whether IgM in the patient contributed to the carbohydrate antigen 19-9 immunoassay, immunoprecipitation was performed. Furthermore, mouse immunoglobulins were added to the patient's serum to verify that the patient's IgM reacted with it. RESULTS: The carbohydrate antigen 19-9 concentration was >400 and 9.5 kU/L using an AIA 1800 analyser and using a UniCel Dxl 800, respectively. In the single carbohydrate antigen 19-9 peak, the molecular weight corresponded to IgM by size-exclusion high-performance liquid chromatography on a Superose 12 column. In the immunoprecipitation reaction and addition of mouse immunoglobulins, there was interference for anti-human IgM and mouse immunoglobulins whose recoveries were 3.2 and 14.2%, respectively. These results indicated that IgM in the patient's serum interfered with the carbohydrate antigen 19-9 immunoassay using an AIA 1800 analyser. CONCLUSION: A novel transient human anti-mouse antibody generated with immune activation in a carbohydrate antigen 19-9 immunoassay using an AIA 1800 analyser was identified in a patient with rectal cancer after surgical resection. These findings demonstrate the importance of monitoring tumour markers in patients after treatment with mouse monoclonal antibody.

Heat Resistant Characteristics of Major Royal Jelly Protein 1 (MRJP1) Oligomer.

Soluble royal jelly protein is a candidate factor responsible for mammiferous cell proliferation. Major royal jelly protein 1 (MRJP1), which consists of oligomeric and monomeric forms, is an abundant proliferative protein in royal jelly. We previously reported that MRJP1 oligomer has biochemical heat resistance. Therefore, in the present study, we investigated the effects of several heat treatments (56, 65 and 96°C) on the proliferative activity of MRJP1 oligomer. Heat resistance studies showed that the oligomer molecular forms were slightly maintained until 56℃, but the molecular forms were converted to macromolecular heat-aggregated MRJP1 oligomer at 65℃ and 96℃. But, the growth activity of MRJP1 oligomer treated with 96°C was slightly attenuated when compared to unheated MRJP1 oligomer. On the other hand, the cell proliferation activity was preserved until 96℃ by the cell culture analysis of Jurkat cells. In contrast, those of IEC-6 cells were not preserved even at 56°C. The present observations suggest that the bioactive heat-resistance properties were different by the origin of the cells. The cell proliferation analysis showed that MRJP1 oligomer, but not MRJP2 and MRJP3, significantly increased cell numbers, suggesting that MRJP1 oligomer is the predominant proliferation factor for mammiferous cells.

Diverse CD36 expression among Japanese population: defective CD36 mutations cause platelet and monocyte CD36 reductions in not only deficient but also normal phenotype subjects.

INTRODUCTION: CD36 is a multifunctional glycoprotein expressed on various human cells, including platelets and monocytes. Five CD36 gene mutations (C268T, 949insA, 329-339del, 1228-1239del and 629-631del/insAAAAC) are mainly responsible for CD36-deficient phenotypes in Japan. It has also been reported that platelet CD36 expression varies widely among normal phenotype individuals. Here, in order to obtain further insight into CD36 expression, we investigated the association between platelet and monocyte CD36 expression levels and defective mutations in the Japanese population. MATERIALS AND METHODS: Blood samples were collected from 135 healthy Japanese volunteers. CD36 expression levels on platelets and monocytes were quantitatively analyzed by flow cytometry. Real-time PCR, PCR-RFLP and allele-specific PCR were performed to detect mutant genotypes. RESULTS: In this population, we found 2 (1.5%) and 9 (6.7%) CD36-deficient subjects as type I and type II, respectively. Among normal phenotype subjects, CD36 expression levels ranged from 1,259 to 11,002 (4,487±2,017) molecules/platelet and from 211 to 5,150 (1,628±986) molecules/monocyte. Genotyping assay showed that heterozygotes with the defective mutations were present in normal (12.9%) and type II-deficient (66.7%) subjects, and that these heterozygous mutations led to decreases in CD36 surface expression on platelets and monocytes. CONCLUSIONS: Heterozygous CD36 mutations, previously known to lead to deficiency in this molecule, are one of the factors responsible for the diversity of CD36 surface expression levels on platelets and monocytes in normal phenotype subjects.

Laboratory and clinical features of abnormal macroenzymes found in human sera.

We report the analysis of unusual macroenzymes, performed in our laboratory, and review the relevant literature. In particular, we focused on macro AST, macroamylase, macro LD and macro CK. Macroenzymes are seen in healthy subjects, but can also be related to disease; thus, accurate detection is useful in day-to-day clinical practice. The macroenzyme is thought to be a specific antigen-antibody complex from the following findings: (1) the complex could be dissociated under acidic pH levels; (2) binding specificity of immunoglobulin in the complex was observed; (3) the binding site of immunoglobulin in the complex was Fab portion; and (4) the maternal IgG involved with macroenzyme was transferred to her children. This article is part of a Special Issue entitled: Medical Proteomics.

Identification of IgG-κ type macroprolactin found in the serum of an 8-year-old girl.

BACKGROUND: We report a case of macroprolactin (macroPRL) in an 8-year-old girl complaining of an enlarged and painful breast who showed elevated PRL levels by enzyme immunoassay. METHODS: Size-exclusion high-performance liquid chromatography (HPLC) was carried out on a Superose 12 column to estimate the PRL molecular profile. To identify the type of immunoglobulin bound to PRL, immunoprecipitation reactions were performed using three types of anti-heavy chain antibody and two types of anti-light chain. RESULTS: PRL in the patient's serum was eluted at a size slightly larger than the IgG peak by size-exclusion HPLC on a Superose 12 column. In the immunoprecipitation reaction, the PRL concentration of the supernatant mixed with anti-gamma heavy chain and anti-κ light chain was dramatically decreased compared to other types of antiserum. These results indicated that macromolecular PRL was composed of PRL and IgG-κ. CONCLUSION: A rare case of PRL-IgG-κ-type complex was identified in the serum of the girl and the type of immunoglobulin light chain was determined for the first time. When encountering a child with hyperprolactinemia, pediatricians should take into account the possibility of macroPRL to avoid unnecessary investigation and/or treatment.

A sudden onset and the spontaneous remission of severe hypo-high-density lipoprotein cholesterolemia without serious underlying disease: a case report.

BACKGROUND: Severe hypo-high-density lipoprotein (HDL) cholesterolemia is defined by serum values less than 20mg/dl. Few acquired cases, without serious underlying disease, have been reported. CASE: An asymptomatic 75-y-old man was admitted for evaluation of low serum HDL-cholesterol (HDL-C) levels (2-8 mg/dl). The record of periodic medical examinations revealed that a sudden decrease had occurred 5 y ago. Mild anemia and proteinuria were noted but the liver and thyroid function tests were normal. ß-Quantification revealed a relatively low HDL-C (10.8 mg/dl) and the serum lecithin cholesterol acyltransferase (LCAT) activity was low (29.4 nmol/ml/h). Unexpectedly, serum HDL-C levels recovered 2 y after hospital discharge. In addition, the serum LCAT activity, hemoglobin concentrations, and urine protein tests all returned to within the reference interval. Subsequent examinations could not clarify the cause of the sudden onset and spontaneous recovery of the extremely low HDL-C. CONCLUSIONS: We describe an unusual case of acquired HDL-C deficiency in a 75-y-old man that did not have serious pre-existing disease. Recently, extremely low HDL-C levels in patients with the nephrotic syndrome, associated with acquired LCAT deficiency, have been reported. The present case might illustrate a milder form of this disorder, because the clinical findings show many similarities.

Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity.

Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.

BDNF, produced by a TPO-stimulated megakaryocytic cell line, regulates autocrine proliferation.

While human platelets release endogenous brain-derived neurotrophic factor (BDNF) upon activation, a previous report on MEG-01, a megakaryocytic cell line, found no trace of BDNF production, and the pathophysiological function of platelet BDNF has remained elusive. In the present study, we demonstrate that MEG-01 produces BDNF in the presence of TPO and that this serves to potentiate cell proliferation. Our in vitro findings suggest that BDNF regulates MEG-01 proliferation in an autocrine manner, and we suggest that BDNF may be a physiological autocrine regulator of megakaryocyte progenitors.

A rapid method to isolate soluble royal jelly proteins.

Soluble royal jelly (RJ) proteins (SRJPs) include the major RJ protein (MRJP) family, which contribute to the physiological actions of RJ. Although SRJPs are prepared using conventional methods involving dialysis and centrifugation, dialysis is a time-consuming process. We have therefore developed a simple method to isolate SRJPs from RJ. This new method produces 20-fold higher levels of SRJPs than that of the conventional procedure; hence, the levels obtained by the new and existing methods were compared. A 1-h ultracentrifugation separated SRJPs in the supernatant into upper, middle and lower layers. Each layer was analyzed by size-exclusion HPLC, SDS-PAGE and 2-DE. The upper and middle layers contained MRJP2 (52 kDa) and MRJP3 (60-70 kDa), while the lower layer contained MRJP1 (290 kDa). In nature, MRJP1 is a monomer and/or oligomer. When the lower layer was analyzed by Superose 12 HPLC, MRJP1 was predominantly an oligomer. Our MRJP isolation method reduces the procedure time by using ultracentrifugation without dialysis to obtain SRJPs and produces layers containing MRJP1 oligomers, MRJP2 and MRJP3.

Release reaction of brain-derived neurotrophic factor (BDNF) through PAR1 activation and its two distinct pools in human platelets.

Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets. Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting. BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 µM PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in α-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to α-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation. In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the α-granules and cytoplasm of human platelets, and only BDNF in α-granules is released through platelet activation.

Molecular characteristics and physiological functions of major royal jelly protein 1 oligomer.

Royal jelly contains numerous components, including proteins. Major royal jelly protein (MRJP) 1 is the most abundant protein among the soluble royal jelly proteins. In its physiological state, MRJP 1 exists as a monomer and/or oligomer. This study focuses the molecular characteristics and functions of MRJP 1 oligomer. MRJP 1 oligomer purified using HPLC techniques was subjected to the following analyses. The molecular weight of MRJP 1 oligomer was found to be 290 kDa using blue native-PAGE. MRJP 1 oligomer was separated into 55 and 5 kDa spots on 2-D blue native/SDS-PAGE. The 55 kDa protein was identified as MRJP 1 monomer by proteome analysis, whereas the 5 kDa protein was identified as Apisimin by N-terminal amino acid sequencing, and this protein may function as a subunit-joining protein within MRJP 1 oligomer. We also found that the oligomeric form included noncovalent bonds and was stable under heat treatment at 56 degrees C. Furthermore, MRJP 1 oligomer dose dependently enhanced and sustained cell proliferation in the human lymphoid cell line Jurkat. In conclusion, MRJP 1 oligomer is a heat-resistant protein comprising MRJP 1 monomer and Apisimin, and has cell proliferation activity. These findings will contribute to further studies analyzing the effects of MRJP 1 in humans.

Sialyl salivary-type amylase associated with ovarian cancer.

BACKGROUND: There have been many reports describing hyperamylasemia, with a salivary-type amylase phenotype, in patients with malignant tumors and/or multiple myelomas. In contrast, we have discovered and characterized a sialyl salivary-type amylase from multiple myeloma and/or lung cancer cells. This paper reports the first association of sialyl salivary-type amylase with ovarian cancer, discovered and characterized using sera from retrospective studies. METHODS: Based on strictly retrospective observation of amylase zymograms, three samples of patients' sera with abnormally fast-migrating isoamylases were detected. Sialyl salivary-type amylase was determined by neuraminidase treatment and reaction with anti-salivary monoclonal antibody, and the extra elution peak of amylase was detected by size-exclusion HPLC analysis. RESULTS: Sialyl salivary-type amylase was detected in the sera of three female patients with ovarian cancer. The ratio of S3 to S2 sub-band in isoamylase electrophoresis, was slightly over 1.00 in two cases and below 1.00 in the other. These cases were not recognized in routine isoamylase electrophoretic analyses, because the abnormal patterns were weak. CONCLUSION: Sialyl salivary-type amylase was characterized for the first time in the sera of patients with ovarian cancer.

Multiple myeloma associated with sialyl salivary-type amylase.

BACKGROUND: There have been several reports describing a notable hyperamylasaemia in patients with multiple myeloma. Such amylase-producing myelomas have been mainly described in the context of concomitant salivary-type hyperamylasaemia, with sialyl salivary-type amylase identified in a portion of those cases. We investigated the incidence of the production of sialyl salivary-type amylase in serum of multiple myeloma patients. METHODS: Eleven patients (6 male and 5 female) who had been diagnosed as having multiple myeloma were enrolled in this study. Sialyl salivary-type amylase was detected by isoamylase electrophoresis and HPLC analysis, and identified by detecting either abnormal neuraminidase-sensitive band through isoamylase electrophoresis or abnormal extra-elution peak of amylase by means of HPLC analysis. RESULTS: Sialyl salivary-type amylase was detected in 7 out of 11 (63.6%) patients. Median total amylase activity was 154 U/l (range 109-43020). Isoamylase electrophoretic patterns of patients' serum were normal in 5 patients (71.4%) out of 7 patients and salivary-dominant in 2 (50.0%) out of 4 patients. CONCLUSIONS: We consider that there is no significant relationship between total serum amylase level and amylase isoenzyme pattern in the incidence of production of sialyl salivary-type amylase with multiple myeloma.

Establishment and characterization of amylase-producing lung adenocarcinoma cell line, IMEC-2.

BACKGROUND: Few studies have successfully established an amylase-producing lung cancer cell line or have examined its cytological, biochemical and biological features. PATIENTS AND METHODS: Cancer cells, isolated from pleural effusion using a gradient method, were cultivated. RESULTS: Amylase production from the newly established cell line was confirmed by positive staining for alpha-amylase and increased amylase levels in the culture supernatant. Electron microscopy revealed zymogen granule-like structures. Sialylation of salivary-type amylase was confirmed directly from the cell line by examining the neuraminidase sensitivity and amylase elution profile under high-performance liquid chromatography. Neither EGFR or KRAS mutation were found. CONCLUSION: This cell line offers a useful tool for analyzing the pathogenesis and pathophysiology of amylase-producing lung cancers. Moreover, it might be useful for probing the metastasis and invasiveness of lung cancer cells and for developing an early diagnostic method based on sialylated salivary-amylase production.

[Advancement of platelet activation measurement: focusing on platelet-derived microparticle measurement].

The assessment of the extent of platelet activation in various thrombotic disorders is important to detect thrombotic tendencies and monitor antiplatelet therapy. Various methods (platelet factor 4: PF4, beta-thromboglobulin: beta-TG, CD62P, PAC-1, etc.) are used as markers of platelet activation. Platelet-derived microparticles (PDMP) are submicroscopic vesicles released by platelets during activation, and are used as a marker of platelet activation. PDMP are usually determined by flow cytometry. An enzyme-linked immunosorbent assay (ELISA) method in which PDMP are detected with the antibody of CD42a and CD42b, was recently reported. The PDMP sample for ELISA can be preserved, and standardization is also easy. It is expected to become an evaluation method of platelet function in vivo.

Blood rheology and risk of thromboembolism in adult and climacteric women.

Increased age is associated with an increased thrombosing tendency, especially deep vein thrombosis and pulmonary embolism. We investigated blood flow in women of various age groups and found that blood flow decreases with advancing age. Because decreased blood flow can lead to platelet and leukocyte activation, it could be related to the pathogenesis of venous thromboembolism with age.

[Analysis of the dilution deviation in CA19-9 measurement].

CA19-9 widely used as a tumor marker of the pancreas and a bile duct. There are a number of reports which describes the measured value discrepancies between RIA and non-RIA kits. RIA results also have shown lack of the linearity over 70 U/ml when the samples are diluted. The pH condition at assay reaction for RIA had been suggested as the major reason, it has been denied by the results from the same pH condition at assay reaction used by COBAS CORE CA19-9 EIA II. On the other hand, the lack of RIA antibody titer is indicated for the discordant results by changing the sample volume to reagent volume ratio in the reaction. Our further investigation also indicates that the specific Lewis blood type, i.e. Le (a-b+), shows the linearity issues by RIA. The discrepancies are not caused by the reaction pH, but the amount of the antibody used in the RIA kit is closely associated. Considering the CA19-9 antibody nature used in RIA kit, which covers broad molecular range, users need to pay more attention to setting up each laboratory's measuring range.