Takanori Moriyama

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°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.

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.

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.

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.

The basis examination of leukocyte-platelet aggregates with CD45 gating as a novel platelet activation marker.

Platelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy‐to‐use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte–platelet aggregate formation associated with activated platelets in circulation.

Myeloma cells produce sialyl salivary-type amylase

Figure 1 Immunoelectrophoresis of the patient’s serum. 1, normal serum; 2, patient’s serum; A-WS, antiserum against whole human serum; A-IgG, A, M, anti-immunoglobulin G, A, M serum; A-LØK, anti-light chain k serum; A-LØL, anti-light chain l serum. Monoclonal protein is indicated by the arrow. Masahiko Shigemura*, Takanori Moriyama, Tomoyuki Endo, Hitoshi Shibuya, Haruki Suzuki, Masaharu Nishimura, Hitoshi Chiba and Kazuhiko Matsuno 1 Clinical Laboratory, Hokkaido University Hospital, Sapporo, Japan 2 Division of Laboratory Technology, Department of Health Sciences, School of Medicine, Hokkaido University, Sapporo, Japan 3 The Second Department of Internal Medicine, Hokkaido University Hospital, Sapporo, Japan

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.

Immunochemical characterization of monoclonal protein in the serum of a patient with Waldenström’s macroglobulinemia showing both pyroglobulin and cryoglobulin properties

A patient with Waldenstr]oms macroglobulinemia whose serum demonstrated properties of both pyroglobulin and cryoglobulin was studied. A monoclonal (M) protein in the serum of the patient was identified by immunoelectrophoresis (IEP) and immunofixation electrophoresis (IFE) as IgM-lambda. The M-protein was separated by gel permeation high-performance liquid chromatography (HPLC). Cryoglobulin in the serum was isolated by the method of cold precipitation. The cryoglobulin was identified as IgM-lambda type by IEP and was the same M-protein as that which occurred in this patients serum. The purified cryoglobulin also had the properties of a pyroglobulin. Neither property disappeared following pretreatment with 2-mercaptoethanol (2-ME), urea, and Triton X-100 detergent, and deglycosylation with N-glycanase (E.C. 3.5.1.52). We suggest that these abnormal properties were caused by the molecular abnormality of the IgM-lambda M-protein.

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.