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1.
Anticancer Res ; 44(4): 1575-1582, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38537961

ABSTRACT

BACKGROUND/AIM: Neutrophil-to-lymphocyte ratio (NLR) is a prognostic indicator for several malignancies, including pancreatic cancer. We developed a novel combined NLR score (cNLRS) based on baseline NLR and change in NLR after chemotherapy (ΔNLR), and examined its prognostic value and role in chemotherapeutic response in patients with advanced pancreatic cancer. PATIENTS AND METHODS: This study retrospectively assessed 210 advanced pancreatic cancer patients receiving chemotherapy between 2010 and 2021. The cNLRS was developed and its association with chemotherapeutic response and prognosis was investigated. RESULTS: The cNLRS consisted of baseline NLR ≥2.5 and ΔNLR ≥0, both of which were remained as independent poor predictors of prognosis adjusting for other traditional clinicopathological features. A high cNLRS served as an independent prognostic factor of reduced overall survival. Of note, the cNLRS was significantly associated with disease control rate and treatment duration not only in 1st line treatment but also in 2nd line treatment. CONCLUSION: The cNLRS established as a useful prognostic biomarker might be associated with chemotherapeutic response and could predict survival in advanced patients with pancreatic ductal adenocarcinoma treated with chemotherapy.


Subject(s)
Neutrophils , Pancreatic Neoplasms , Humans , Neutrophils/pathology , Retrospective Studies , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Prognosis , Lymphocytes/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology
2.
Transplant Proc ; 56(1): 239-243, 2024.
Article in English | MEDLINE | ID: mdl-38218698

ABSTRACT

Liver transplantation (LT) is the only life-saving option when acute-on-chronic liver failure (ACLF) does not improve with conservative therapy. Acute pancreatitis (AP) can cause chronic liver disease progression to ACLF. However, deceased donor LT for patients with AP has had mixed results, and no consensus has been established regarding the indication for LT. We report the first successful living donor LT (LDLT) for ACLF caused by severe AP. The 38-year-old patient with alcoholic liver disease was transferred to our institute with worsening refractory ascites. During the pretransplant workup, she developed severe acute necrotizing pancreatitis, resulting in grade 3 ACLF. The patient's clinical course was further complicated by high levels of donor-specific antibodies and immune thrombocytopenia. The AP gradually improved after intensive care combined with artificial liver support. The patient successfully underwent urgent LDLT with upfront splenectomy and desensitization therapy, including plasm exchange, high-dose intravenous immunoglobulin, and anti-thymocyte globulin. No infection or recurrence of AP was observed postoperatively. We conclude that LDLT is a feasible option for ACLF patients caused by severe AP if a deceased donor is not readily available.


Subject(s)
Acute-On-Chronic Liver Failure , Liver Transplantation , Pancreatitis, Acute Necrotizing , Female , Humans , Adult , Liver Transplantation/adverse effects , Liver Transplantation/methods , Acute-On-Chronic Liver Failure/etiology , Acute-On-Chronic Liver Failure/surgery , Living Donors , Pancreatitis, Acute Necrotizing/complications , Pancreatitis, Acute Necrotizing/surgery , Acute Disease , Retrospective Studies
3.
Surg Open Sci ; 16: 215-220, 2023 Dec.
Article in English | MEDLINE | ID: mdl-38035224

ABSTRACT

Background: The principle of hepatoblastoma (HB) treatment is complete resection. The removal of tumor-bearing section(s) or hemiliver is widely accepted. However, neither the standardized anterior approach for right hepatectomy nor parenchymal sparing anatomical liver resection has been described for HB. Methods: We retrospectively reviewed the clinical course of two pediatric HB patients who underwent extended right hepatectomy using the anterior approach with the liver hanging maneuver and one who underwent parenchymal sparing anatomical liver resection of S4 apical+S8 ventral/dorsal+S7. The critical aspects of surgical techniques are described in detail. Results: In all three patients, R0 resection was achieved without complications and are currently alive without recurrence after an average follow-up of 23 months. Intraoperative cardiac hemodynamics were stable, even in a trisomy 18 patient with cardiac disease. Conclusions: Our findings suggest that these innovative techniques established in adults are safe and feasible for HB in children. These techniques also allow optimal anatomical liver resection to accomplish curative surgery while maintaining the functional reserve of the remnant liver.

4.
Asian J Endosc Surg ; 16(3): 546-549, 2023 Jul.
Article in English | MEDLINE | ID: mdl-36944530

ABSTRACT

Although laparoscopic cholecystectomy is a well-established surgical procedure, an accessory hepatic duct (AcHD) entering the cystic duct is poorly understood. A 77-year-old woman with symptomatic cholecystlithiasis was referred to our hospital. Abdominal ultrasonography indicated several small stones in the gall bladder. Magnetic resonance cholangiopancreatography (MRCP) did not reveal an anomalous cystic duct. Dissecting the gall bladder bed at operation, AcHD entering the cystic duct was suspected. Intraoperative cholangiography revealed that B5 branch entered the cystic duct. We ligated the AcHD, and divided it. Laparoscopic cholecystectomy was completed, and the patient was discharged without any complication. A week after the operation, MRCP showed that ventral branch of B5 was dilated. The patient showed no symptom for more than a year. The present case exhibited extremely rare AcHD entering the cystic duct, which was hardly recognized before surgery. It is possible to recognize such anomalous variants with standard laparoscopic approach based on 2018 Tokyo Guidelines and with attention to the possibilities of AcHD entering the cystic duct.


Subject(s)
Cholecystectomy, Laparoscopic , Cholecystolithiasis , Female , Humans , Aged , Cystic Duct/surgery , Cholecystectomy, Laparoscopic/methods , Cholecystolithiasis/complications , Cholecystolithiasis/surgery , Hepatic Duct, Common/surgery , Cholangiography
5.
J Immunol Methods ; 511: 113384, 2022 12.
Article in English | MEDLINE | ID: mdl-36372268

ABSTRACT

In general, it is difficult to raise novel monoclonal antibodies against relatively low-molecular weight antigen, and particularly those with high homology for the mouse protein. The optimized B-cell targeting (BCT) technique can overcome this limitation. The point of this advanced technology is the selection of sensitized B lymphocytes by the antigen through B-cell receptors (BCRs). This strict selection by specific and strong interaction between antigen and antibody enables the efficient production of monoclonal antibodies with high specificity and affinity. It also offers the condensation of sensitized target B lymphocytes to selectively generate hybridoma cells secreting desired monoclonal antibodies. In this study, several kinds of biotinylated human myoglobin (hMyo) were prepared to select sensitized B lymphocytes via BCRs. Biotinylated hMyo prepared by a 3.75- and 7.5-fold molar excess of N-hydroxysuccinimide (NHS)-biotin provided high antigenicity of 68-88%. B lymphocytes selected by these biotinylated antigens had an ELISA-positive rate >17 times higher than that with usual biotinylated antigen. Monoclonal antibodies generated by the optimized BCT technology by preselecting sensitized B lymphocytes with the target antigen were identified to specifically recognize lower antigenic epitopes in hMyo with high affinity, while this would be impossible by the polyethylene glycol (PEG) method. Furthermore, combination of these high-affinity monoclonal antibodies gave the best binding rate in an epitope binning assay. These outcomes could be attributed to the unique characteristic that BCRs on sensitized B lymphocytes themselves can select the target epitopes in the antigen. The BCRs may act as a strict sensor of B lymphocytes to precisely select the target epitopes, even though the number of immunized B lymphocytes is low.


Subject(s)
Antibodies, Monoclonal , Receptors, Antigen, B-Cell , Humans , Animals , Mice , Technology
6.
Anal Sci ; 38(2): 235-239, 2022 Feb.
Article in English | MEDLINE | ID: mdl-35286647

ABSTRACT

This paper reports a superiority of the asymmetric electric field formed in the rectangle microwell array for the electrofusion of splenocytes and myeloma cells with different diameters. The upper substrate with microband electrodes was mounted on the lower substrate with the microwell array. Two electrodes were arranged at the both sides of the microwells on the bottom surface. An attractive force of positive dielectrophoresis was employed to capture splenocytes with smaller diameter and myeloma cells with larger diameter at the right and left of microwells by applying AC electric field. The splenocytes and myeloma cells were fused by the asymmetric electric field that was generated in the microwells by applying DC electric pulse to the bottom electrode at the right side. The asymmetric field could allow to the formation of small openings on the membrane for the fusion of smaller splenocytes by experiencing higher field and the suppression for the disruption of larger myeloma cells by experiencing lower field.


Subject(s)
Electricity , Electrodes
7.
Ann Vasc Dis ; 15(4): 308-316, 2022 Dec 25.
Article in English | MEDLINE | ID: mdl-36644254

ABSTRACT

Objectives: This study aims to discuss the midterm results of thoracic endovascular aortic repair (TEVAR) with reentry closure for chronic type B aortic dissection (CTBAD). Materials and Methods: This retrospective study analyzed 13 patients with CTBAD who underwent TEVAR with reentry closure between July 2014 and December 2020. We evaluated the false lumen (FL) cross-sectional area using computed tomography images of the descending aorta at the level of the bronchial bifurcation, Valsalva sinus, celiac artery, and infrarenal abdominal aorta pre- and postoperation. The study endpoints were technical and clinical success rates, freedom from additional aortic reintervention or surgery, and survival. Results: Technical success was obtained in 12 patients (92.3%) with no hospital mortality and neurological complications. The postoperative observation period was 49.2±21.5 months. The clinical success rate was 76.9% (10 cases), and a postoperative reduction of the FL cross-sectional area was obtained in 53.8% of patients. The 5-year overall survival rate was 64.8% with no aortic-related deaths while the 5-year freedom from additional aortic surgery rate was 66.7%. Conclusions: TEVAR with reentry closure suggests preventing FL dilatation or rupture in CTBAD, but the revision of our devices and further research with more patients and longer follow-up periods are required.

8.
Ann Vasc Dis ; 15(4): 341-343, 2022 Dec 25.
Article in English | MEDLINE | ID: mdl-36644269

ABSTRACT

Congenital abdominal aortic aneurysm (AAA) with coarctation has been considered an extremely rare condition. In this study, we present a 3-year-old boy, who was diagnosed by chance with congenital AAA at first operation. We replaced the AAA+coarctation with a 6-mm polytetrafluoroethylene (PTFE) graft. Histological examination of the aortic wall revealed no particular abnormalities. Collateral vessels were noted to develop over 14 years of followup. Good blood flow to both lower limbs and no intermittent claudication were observed. After growth, at the age 17, he underwent extra-anatomical bypass using a 12-mm PTFE graft. This is the first report of successful treatment of congenital AAA+coarctation with longterm followup.

9.
Int Immunopharmacol ; 98: 107872, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34182241

ABSTRACT

It is quite difficult to generate monoclonal antibodies that recognize the three-dimensional structures of the antigens of interest. To address this limitation, we developed a new hybridoma technology termed "optimized stereospecific targeting (SST)". Here we aimed at generating stereospecific monoclonal antibodies against a G protein-coupled receptor (GPCR). The optimized SST technique enabled the efficient production of conformation-specific monoclonal antibodies against human corticotropin-releasing hormone receptor 1 (huCRHR1). Hybridoma cells secreting stereospecific monoclonal antibodies were selectively cloned by a limiting dilution method and the target monoclonal antibodies were purified by protein A column chromatography. They specifically cross-reacted with native huCRHR1 expressed on the surface of CHO cells, whereas they showed no affinity for MDA-MB-231 cancer cells, which abundantly express EphA2 on the cell surface. Furthermore, immunofluorescence analysis revealed that treatment of huCRHR1-expressing CHO cells with 4% paraformaldehyde led to a decrease in the affinity of purified monoclonal antibodies for intact huCRHR1 on the cell surface. In addition, purified monoclonal antibodies showed no cross-reactivity with huCRHR1 expressed on Sf9 insect cells. These results strongly suggest that monoclonal antibodies generated by the optimized SST technique feature specific binding to the intact form of the target GPCR on mammalian cells.


Subject(s)
Antibodies, Monoclonal/pharmacology , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/therapeutic use , Antibody Specificity , CHO Cells , Cell Line, Tumor , Cricetulus , Cross Reactions , Female , Humans , Mice , Receptor, EphA2/metabolism , Receptors, Corticotropin-Releasing Hormone/genetics , Receptors, Corticotropin-Releasing Hormone/ultrastructure , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Sf9 Cells , Spodoptera
10.
J Immunol Methods ; 484-485: 112813, 2020.
Article in English | MEDLINE | ID: mdl-32592774

ABSTRACT

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.


Subject(s)
Antibodies, Monoclonal/immunology , Breast Neoplasms/immunology , Ephrin-A2/immunology , Immunoglobulin G/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibody Specificity , B-Lymphocytes/immunology , Breast Neoplasms/chemistry , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , CHO Cells , Cell Line, Tumor , Cricetulus , Enzyme-Linked Immunosorbent Assay , Ephrin-A2/chemistry , Ephrin-A2/genetics , Ephrin-A2/metabolism , Female , Fluorescent Antibody Technique , Humans , Hybridomas , Immunization , Immunoglobulin G/biosynthesis , Mice , Mice, Inbred BALB C , Protein Conformation , Receptor, EphA2 , Receptors, Antigen, B-Cell/immunology , Structure-Activity Relationship
11.
Transgenic Res ; 29(3): 339-353, 2020 06.
Article in English | MEDLINE | ID: mdl-32367383

ABSTRACT

Fibrinogen from human blood is used as a main component of coagulants, including surgical tissue sealants. The development of a recombinant human fibrinogen (rFib) is anticipated to eliminate the risks of blood-borne infections. Here, we report the efficient production of rFib in a transgenic silkworm system. A silkworm line carrying cDNAs of the fibrinogen Aα and γ chains (Aα/γ-silkworm) produced Aα and γ chains in its cocoons, however, the Bß chains were not detected from cocoons of another silkworm line carrying the cDNA of fibrinogen Bß chains (Bß-silkworm). A silkworm line for all three fibrinogen chains was generated by crossing Aα/γ-silkworms with Bß-silkworms, which secreted Aα2Bß2γ2 fibrinogen (rFib) into cocoons at high contents. The N-terminal amino acid sequences of the three rFib chains were identical to those of the corresponding chains of native fibrinogen (nFib). The N-glycan profile of the rFib comprised oligomannose-type (53%), complex-type (34%), and paucimannose-type (13%); neither high-mannose-type (six or more mannose residues) nor core-fucosylated glycans were observed. The coagulation activity of the rFib was evaluated for the amount of thrombin-released fibrinopeptide A (FpA) and the kinetics for turbidity increase (non-covalent network formation) in the solution. FpA release rates were equivalent between rFib and nFib; by contrast, the kinetics of the turbidity increase for rFib were accelerated nearly two-fold, for both the rate and maximum value, compared to those of nFib. These results demonstrate that the rFib produced in the transgenic silkworm system is comparable to nFib in both physical and coagulative properties. This rFib is a promising candidate component for safe hemostatic pharmaceuticals.


Subject(s)
Animals, Genetically Modified/metabolism , Fibrinogen/metabolism , Recombinant Proteins/metabolism , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/growth & development , Bombyx , Fibrinogen/genetics , Glycosylation , Humans , Recombinant Proteins/genetics
12.
Biosci Biotechnol Biochem ; 84(4): 686-694, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31852366

ABSTRACT

Budded viruses (BVs) of baculovirus such as Autographa californica nucleopolyhedrovirus (AcNPV) have recently been studied as biological nanomaterials, and methods for their longer-term storage without deterioration would be desirable. The cryopreservation of virions with a naturally occurring saccharide like trehalose as a cryoprotectant is known to be useful for maintaining the viral structure and function. In this study, we examined how useful trehalose is as protectant for BV cryopreservation during repeated freeze-thaw cycles: 1) membrane fusion between liposomes (multilamellar vesicles, MLVs) and BVs, 2) infection of insect culture cells (Sf9 cells) by RFP-expressing BVs, and 3) morphologies of these BVs were investigated by fluorescent dequenching assay, fluorescence microscopy, and transmission electron microscopy (TEM), respectively. The results suggest that the BVs deteriorate in quality with each freeze-thaw cycle, and this deterioration can be diminished with the use of trehalose to an extent similar to that seen with storage on ice.


Subject(s)
Cryoprotective Agents/pharmacology , Freezing , Membrane Fusion , Nucleopolyhedroviruses/pathogenicity , Trehalose/pharmacology , Virion/physiology , Animals , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Sf9 Cells
13.
J Biosci Bioeng ; 128(5): 578-584, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31147218

ABSTRACT

Until now, various kinds of monoclonal antibodies have been raised against many antigens. Nevertheless, the production of these monoclonal antibodies was usually limited to only one antigen. If simultaneous generation of monoclonal antibodies against multiple antigens were available at one time, we could reduce not only laborious work, but also experimental animals. Here, we developed a multitargeting (MT) method that enables simultaneous production of monoclonal antibodies against multiple antigens on the basis of strict selection of sensitized B lymphocytes by the target antigens via B-cell receptors. After immunization using multiple antigens, monoclonal antibodies against four different antigens containing lower antigenic one were successfully generated only in one experiment. At maximum, more than 90 % of ELISA-positive wells to hybridoma-positive ones was obtained by this advanced technology. This must be attributed to strict selection of sensitized B lymphocytes by different antigens. In the MT method, sensitized B lymphocytes were selected by means of each desired antigen regardless of their antigenic differences. Selective fusion of B cell-myeloma cell complexes by electrical pulses was also of critical importance for efficient generation of hybridoma cells secreting desired monoclonal antibodies. This study strongly suggests that simultaneous production of novel monoclonal antibodies directed against multiple antigens of interest by the MT method can be feasible.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Receptors, Antigen, B-Cell/immunology , Animals , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Enzyme-Linked Immunosorbent Assay , Hybridomas/immunology
14.
Anal Sci ; 35(6): 701-704, 2019 Jun 10.
Article in English | MEDLINE | ID: mdl-30773512

ABSTRACT

We have developed a simple and rapid formation of a cell-based array on microwell array electrodes by an attractive force of positive dielectrophoresis (p-DEP), even after removing an upper disk electrode stick that was used as a counter electrode to the microwell array electrodes. The attractive force of p-DEP generated by the scanning of the disk electrode allows the formation of a cell-based array on all microwell arrays. We demonstrated an exploration of target cells spiked with a low ratio after removing the disk electrode.

15.
Immunotherapy ; 11(2): 119-127, 2019 02.
Article in English | MEDLINE | ID: mdl-30730271

ABSTRACT

Attention to therapeutic monoclonal antibodies has been dramatically increasing year by year. Their highly specific targeting of antigens can provide very effective medical treatment, and the advent of molecular-targeting medicine is allowing development of a new generation of therapeutic agents. However, there is one critical obstacle to overcome. Most of the established therapeutic monoclonal antibodies have specificity for the primary structures of target antigens, although all proteins harbor original native intact structures for their own specific functions. Stereo-specific monoclonal antibodies recognizing conformational structures of target antigens may thus offer a markedly more versatile approach. Their application may change the very concepts underlying use of therapeutic antibodies.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Autoimmune Diseases/therapy , Dermatitis, Atopic/therapy , Immunotherapy/methods , Neoplasms/therapy , Animals , Antibody Affinity , Autoimmune Diseases/immunology , Dermatitis, Atopic/immunology , Drug Approval , Epitopes, B-Lymphocyte/immunology , Humans , Neoplasms/immunology , Protein Conformation , Stereoisomerism
16.
BMC Vet Res ; 14(1): 260, 2018 Aug 31.
Article in English | MEDLINE | ID: mdl-30170576

ABSTRACT

BACKGROUND: The generation of recombinant proteins for commercialisation must be cost-effective. Despite the cost-effective production of recombinant feline interferon (rFeIFN) by a baculovirus expression system, this rFeIFN carries insect-type N-glycans, with core α 1,3 fucosyl residues that act as potential allergens. An alternative method of production may yield recombinant glycoproteins with reduced antigenicity. RESULTS: A cDNA clone encoding the fifteenth subtype of FeIFN-α (FeIFN-α15) was isolated from a Japanese domestic cat. This clone encoded a protein of 189 amino acids with a molecular mass of 21.1 kDa. The rFeIFN-α15 was expressed using a transgenic silkworm system, which was expected to yield an N-glycan structure with reduced antigenicity compared with the protein produced by the baculovirus system. The resulting rFeIFN-α15 accumulated in the sericin layer of silk fibres and was easily extracted and purified by column chromatography. The N-terminal amino acid sequence of purified rFeIFN-α15 was identical to the mature form of natural sequence. Moreover, its N-glycans did not include detectable core α 1,3 fucosyl residues. Its anti-vesicular stomatitis virus activity (2.6 × 108 units/mg protein) was comparable to that of the baculovirus-expressed rFeIFN. CONCLUSIONS: The lower allergy risk of rFeIFN produced by the transgenic silkworm system than by the baculovirus expression system is due to the former lacking core α 1,3 fucosyl residues in its N-glycans. The rFeIFN-α15 produced by the transgenic silkworm system may be a prospective candidate for the next generation of rFeIFN in veterinary medicine.


Subject(s)
Bombyx/metabolism , Interferons/biosynthesis , Polysaccharides/chemistry , Amino Acid Sequence , Animals , Animals, Genetically Modified , Bombyx/genetics , Cats , Interferons/genetics , Interferons/immunology , Polysaccharides/genetics , Polysaccharides/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Silk/chemistry
17.
Yakugaku Zasshi ; 138(7): 875-884, 2018.
Article in Japanese | MEDLINE | ID: mdl-29962463

ABSTRACT

 There exists an increasing need to produce useful proteins in recombinant technologies. In particular, most biologics for medical purposes are produced as recombinant proteins. Various host cell/vector systems have been developed, but it remains difficult to efficiently produce large molecular weight proteins with complex structures. As a result of breeding for several thousand years, the silkworm has acquired the ability to synthesize bulk amounts of silk proteins. To utilize this capacity for the mass production of useful proteins, transgenic silkworms have been generated that synthesize recombinant proteins in the silk gland and secrete them into the silk cocoon. Using this transgenic silkworm system, various proteins, including antibodies, collagen, and fibrinogen, have been successfully produced and are being developed as materials for diagnostic or research-use reagents, as well as for cosmetics. Moreover, several silkworm-produced proteins are being developed as biologics for therapeutic use. Transgenic silkworms need to be reared under good manufacturing practices (GMP)-compliant conditions to produce biologics. Therefore, we have constructed a GMP-compliant pilot plant for producing biologics using transgenic silkworm, and are now developing silkworm-rearing technology under GMP-compliant conditions.


Subject(s)
Animal Husbandry/methods , Animals, Genetically Modified , Biological Products , Bombyx , Animals , Antibodies , Bombyx/metabolism , Collagen , Cosmetics , Fibrinogen , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism
18.
Biochem Biophys Res Commun ; 501(4): 982-987, 2018 07 02.
Article in English | MEDLINE | ID: mdl-29775614

ABSTRACT

Close homolog of L1 (CHL1) and its truncated form mainly play crucial roles in mouse brain development and neural functions. Herein, we newly identified that truncated form of CHL1 is produced and released from lung tumor tissue in a mouse model expressing human EML4-ALK fusion gene. Both western blot and direct ELISA analysis revealed that mouse CHL1 level in serum (including serum extracellular vesicles) was significantly elevated in EML4-ALK transgenic mice. The correlation between the tumor size and the amount of CHL1 secretion could be examined in this study, and showed a significant positive correlation in a tumor size-dependent manner. Considering these results, the measurement of circulating CHL1 level may contribute to assess a tumor progression in human lung tumor patients.


Subject(s)
Cell Adhesion Molecules/blood , Cell Adhesion Molecules/metabolism , Lung Neoplasms/blood , Animals , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , HEK293 Cells , Humans , Lung Neoplasms/pathology , Mice, Inbred C57BL , Tumor Burden
19.
Colloids Surf B Biointerfaces ; 155: 248-256, 2017 Jul 01.
Article in English | MEDLINE | ID: mdl-28432958

ABSTRACT

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases.


Subject(s)
Baculoviridae/chemistry , Proteolipids/chemistry , Receptors, Corticotropin-Releasing Hormone/chemistry , Unilamellar Liposomes/chemistry , Viral Envelope Proteins/chemistry , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Humans , Hydrogen-Ion Concentration , Luminescent Proteins/chemistry , Membrane Fusion , Recombinant Proteins/chemistry , Rhodamines/chemistry , Solutions , Spectrometry, Fluorescence , Red Fluorescent Protein
20.
Molecules ; 21(12)2016 Dec 11.
Article in English | MEDLINE | ID: mdl-27973427

ABSTRACT

3-Mercaptopyruvate sulfurtransferase (MST) is one of the principal enzymes for the production of hydrogen sulfide and polysulfides in mammalians, and emerging evidence supports the physiological significance of MST. As a fundamental study of the physiology and pathobiology of MST, it is necessary to establish the tissue distribution of MST in mice. In the present study, the expression of MST in various organs of adult and fetal mice was analyzed by Western blotting and enzyme-immunohistochemistry. Moreover, the histology of MST gene-deficient mice was examined. Western blotting revealed that all organs examined had MST. The brain, liver, kidneys testes, and endocrine organs contained large amounts of MST, but the lungs, spleen, thymus, and small intestine did not. Immunohistochemically, the MST expression pattern varies in a cell-specific manner. In the brain, neural and glial cells are positively stained; in the lung, bronchiolar cells are preferentially stained; in the liver, hepatocytes around central veins are more strongly stained; renal convoluted cells are strongly stained; and pancreatic islets are strongly stained. Fetal tissues were studied, and MST expression was found to be similar before and after birth. Histological observation revealed no remarkable findings in MST gene-deficient mice. The present study revealed fundamental information regarding the MST expression of various organs in adult and fetal mice, and the morphological phenotype of MST gene-deficient mice.


Subject(s)
Brain/metabolism , Bronchioles/metabolism , Fetus/metabolism , Islets of Langerhans/metabolism , Liver/metabolism , Sulfurtransferases/biosynthesis , Sulfurtransferases/genetics , Animals , Brain/cytology , Hepatocytes/metabolism , Hydrogen Sulfide/metabolism , Liver/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuroglia/metabolism , Neurons/metabolism
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