Publications by authors named "Shiho Tanaka"

56 Publications

Rapid Identification of Neutralizing Antibodies against SARS-CoV-2 Variants by mRNA Display.

bioRxiv 2021 Sep 14. Epub 2021 Sep 14.

The increasing prevalence of SARS-CoV-2 variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly-reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identified a set of antibodies against SARS-CoV-2 spike (S) proteins and characterized the structures of nAbs that recognized epitopes in the S1 subunit of the S glycoprotein. These structural studies revealed distinct binding modes for several antibodies, including targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interacts with angiotensin- converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. A potent ACE2-blocking nAb was further engineered to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is a promising approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.
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http://dx.doi.org/10.1101/2021.09.14.460356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452091PMC
September 2021

Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice.

Sci Rep 2021 07 21;11(1):14917. Epub 2021 Jul 21.

ImmunityBio, Inc., 9920 Jefferson Blvd, Culver City, CA, 90232, USA.

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity-including mucosal immunity-against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.
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http://dx.doi.org/10.1038/s41598-021-94364-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295250PMC
July 2021

An ACE2 Triple Decoy that neutralizes SARS-CoV-2 shows enhanced affinity for virus variants.

Sci Rep 2021 06 17;11(1):12740. Epub 2021 Jun 17.

ImmunityBio, Inc., 9920 Jefferson Blvd., Culver City, CA, 90232, USA.

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgGF fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.
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http://dx.doi.org/10.1038/s41598-021-91809-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211782PMC
June 2021

Chemical and molecular bases of dome formation in human colorectal cancer cells mediated by sulphur compounds from Cucumis melo var. conomon.

FEBS Open Bio 2020 12 6;10(12):2640-2655. Epub 2020 Nov 6.

Laboratory of Plant Life Science and Technology, United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan.

Colorectal cancer was the third most commonly diagnosed malignant tumor and the fourth leading cause of cancer deaths worldwide in 2012. A human colorectal cancer cell line, RCM-1, was established from a colon cancer tissue diagnosed as a well-differentiated rectum adenocarcinoma. RCM-1 cells spontaneously form 'domes' (formerly designated 'ducts') resembling villiform structures. Two sulphur-containing compounds from Cucumis melo var. conomon (Katsura-uri, or Japanese pickling melon), referred to as 3-methylthiopropionic acid ethyl ester (MTPE) and methylthioacetic acid ethyl ester (MTAE), can induce the differentiation of the unorganized cell mass of an RCM-1 human colorectal cancer cell culture into a dome. However, the underlying molecular mechanisms of such dome formation have not been previously reported. Here, we performed a structure-activity relationship analysis, which indicated that methylthioacetic acid (MTA) was the lowest molecular weight compound with the most potent dome-inducing activity among 37 MTPE and MTAE analogues, and the methylthio group was essential for this activity. According to our microarray analysis, MTA resulted in down-regulation of 537 genes and up-regulation of 117 genes. Furthermore, MTA caused down-regulation of many genes involved in cell-cycle control, with the cyclin E2 (CCNE2) and cell division cycle 25A (CDC25A) genes being the most significantly reduced. Pharmacological analysis showed that the administration of two cell-cycle inhibitors for inactivating CDC25A phosphatase (NSC95397) and the cyclin E2/cyclin-dependent kinase 2 complex (purvalanol A) increased the dome number independently of MTA. Altogether, our results indicate that MTA is the minimum unit required to induce dome formation, with the down-regulation of CDC25A and possibly CCNE2 being important steps in this process.
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http://dx.doi.org/10.1002/2211-5463.13001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714076PMC
December 2020

Deep learning-based classification of rectal fecal retention and analysis of fecal properties using ultrasound images in older adult patients.

Jpn J Nurs Sci 2020 Oct 11;17(4):e12340. Epub 2020 May 11.

Global Nursing Research Center, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Aim: The present study aimed to analyze the use of machine learning in ultrasound (US)-based fecal retention assessment.

Methods: The accuracy of deep learning techniques and conventional US methods for the evaluation of fecal properties was compared. The presence or absence of rectal feces was analyzed in 42 patients. Eleven patients without rectal fecal retention on US images were excluded from the analysis; thus, fecal properties were analyzed in 31 patients. Deep learning was used to classify the transverse US images into three types: absence of feces, hyperechoic area, and strong hyperechoic area in the rectum.

Results: Of the 42 patients, 31 tested positive for the presence of rectal feces, zero were false positive, zero were false negative, and 11 were negative, indicating a sensitivity of 100% and a specificity of 100% for the detection of rectal feces in the rectum. Of the 31 positive patients, 14 had hard stools and 17 had other types. Hard stool was detected by US findings in 100% of the patients (14/14), whereas deep learning-based classification detected hard stool in 85.7% of the patients (12/14). Other stool types were detected by US findings in 88.2% of the patients (15/17), while deep learning-based classification also detected other stool types in 88.2% of the patients (15/17).

Conclusions: The results showed that US findings and deep learning-based classification can detect rectal fecal retention in older adult patients and distinguish between the types of fecal retention.
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http://dx.doi.org/10.1111/jjns.12340DOI Listing
October 2020

The human IL-15 superagonist N-803 promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-suppressed, SHIV-infected macaques.

PLoS Pathog 2020 03 12;16(3):e1008339. Epub 2020 Mar 12.

Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, United States of America.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.
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http://dx.doi.org/10.1371/journal.ppat.1008339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093032PMC
March 2020

A PQ-loop protein Ypq2 is involved in the exchange of arginine and histidine across the vacuolar membrane of Saccharomyces cerevisiae.

Sci Rep 2019 10 21;9(1):15018. Epub 2019 Oct 21.

Department of Bioscience, Graduate school of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan.

In nutrient-rich conditions, basic amino acids are actively accumulated into the vacuoles by H-coupled transporters in Saccharomyces cerevisiae. In addition to the H-coupled systems, the existence of an exchanger for arginine and histidine was indicated by kinetic analysis using isolated vacuolar membrane vesicles; however, the gene(s) involved in the activity has not been identified. Here, we show that the uptake activity of arginine driven by an artificially imposed histidine gradient decreased significantly by the disruption of the gene encoding vacuolar PQ-loop protein Ypq2, but not by those of Ypq1 and Ypq3. The exchange activity was restored by the expression of YPQ2. Furthermore, the substitution of a conserved proline residue, Pro29, in Ypq2 greatly decreased the exchange activity. These results suggest that Ypq2 is responsible for the exchange activity of arginine and histidine across the vacuolar membrane, and the conserved proline residue in the PQ-loop motif is required for the activity.
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http://dx.doi.org/10.1038/s41598-019-51531-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803629PMC
October 2019

Function and Role of ATP-Binding Cassette Transporters as Receptors for 3D-Cry Toxins.

Toxins (Basel) 2019 02 19;11(2). Epub 2019 Feb 19.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka 2-24-16, Koganei, Tokyo 184-8588, Japan.

When ABC transporter family C2 (ABCC2) and ABC transporter family B1 (ABCB1) were heterologously expressed in non-susceptible cultured cells, the cells swelled in response to Cry1A and Cry3 toxins, respectively. Consistent with the notion that 3D-Cry toxins form cation-permeable pores, ABCC2 (BmABCC2) facilitated cation-permeable pore formation by Cry1A when expressed in oocytes. Furthermore, BmABCC2 had a high binding affinity () to Cry1Aa of 3.1 × 10 M. These findings suggest that ABC transporters, including ABCC2 and ABCB1, are functional receptors for 3D-Cry toxins. In addition, the Cry2 toxins most distant from Cry1A toxins on the phylogenetic tree used ABC transporter A2 as a receptor. These data suggest that 3D-Cry toxins use ABC transporters as receptors. In terms of inducing cell swelling, ABCC2 has greater activity than cadherin-like receptor. The pore opening of ABC transporters was hypothesized to be linked to their receptor function, but this was repudiated by experiments using mutants deficient in export activity. The synergistic relationship between ABCC2 and cadherin-like receptor explains their ability to cause resistance in one species of insect.
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http://dx.doi.org/10.3390/toxins11020124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409751PMC
February 2019

[The evaluation of stored feces in elderly patients by ultrasonography: Three case studies].

Nihon Ronen Igakkai Zasshi 2018 ;55(4):657-662

Global Nursing Research Center, Graduate School of Medicine, The University of Tokyo.

Providing defecation care can be challenging because bowel movements cannot be directly observed in home-care settings, and the objective evaluation of constipation symptoms is difficult, particularly for elderly patients with cognitive impairment. We evaluated the use of rectal ultrasonography (US) to assess the properties and volume of feces in three cases with different fecal properties. Case 1: In a 94-year-old man with normal feces (Bristol stool score: BS type 4), rectal US revealed a crescent-shaped high-echo area without acoustic shadow that was present until the next defecation. Case 2: In a 92-year-old woman with hard stool (BS type 1), rectal US showed a crescent-shaped strong-echo area with acoustic shadow that was present until the next defecation. The length of the high-echo area gradually increased during the observation period and decreased after defecation in Cases 1 and 2. Case 3: In a 67-year-old man with watery stool (BS type 7), rectal US revealed a low-peripheral-frequency-echo area without acoustic shadow. Rectal ultrasonography was able to demonstrate the presence or absence of hard stool, which was observed as a crescent-shaped a strong, high-echo area with acoustic shadow; the presence or absence of hard stool may be evaluated based on these findings. Furthermore, the fecal volume may be able to be evaluated based on the long diameter of the crescent-shaped high-echo area. Determining the best course of defecation care based on the fecal properties/volume evaluated using rectal US will likely be possible in the future.
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http://dx.doi.org/10.3143/geriatrics.55.657DOI Listing
April 2019

Ultrasonographic evaluation of changes over time in one defecation cycle in adults with functional constipation: A report of two cases.

Drug Discov Ther 2018;12(5):304-308

Global Nursing Research Center, Graduate School of Medicine, The University of Tokyo.

We described fecal retention during the defecation cycles of adults with functional constipation via ultrasonography (US) of the large intestine. US was performed continuously after the last defecation until the next defecation. We defined the fecal finding level on US as follows: weak fecal retention, a marginally high echo in the colonic lumen; or strong fecal retention, a strongly echoic colon lumen with showing a crescent-shaped acoustic shadow on transverse images and haustrations on longitudinal images. The findings confirmed weak fecal retention in the colon throughout the defecation cycle and a pattern of strong fecal retention in the descending and sigmoid colon and over the colon, including the transverse colon and ascending colon, in patients with functional constipation.
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http://dx.doi.org/10.5582/ddt.2018.01054DOI Listing
August 2019

Fecal Distribution Changes Using Colorectal Ultrasonography in Older People with Physical and Cognitive Impairment Living in Long-Term Care Facilities: A Longitudinal Observational Study.

Healthcare (Basel) 2018 May 25;6(2). Epub 2018 May 25.

Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1130033, Japan.

Nurses encounter difficulties evaluating constipation in elderly people with physical and cognitive impairment. Transabdominal ultrasonography (US) has been used to evaluate fecal impaction or fecal quality. However, it is unclear whether colorectal US can evaluate constipation symptoms in older people. Using colorectal US, we continuously observed the elderly and clarified the relationship between patterns of fecal distribution changes and constipation symptoms in older people with physical and cognitive impairment at long-term care facilities. This study included patients aged ≥65 years with oral intake. US was performed once a day until the next defecation, and fecal hardness was assessed. US images were extracted and categorized. Then, patterns of fecal distribution changes in the colorectum were classified. Multiple logistic regression analysis was performed to examine related factors associated with a constipation pattern. Among 101 patients, US images of 95 patients were analyzed. In 74.4% of the patients, US showed continuation of reflection with acoustic shadow in the rectum, which was significantly associated with defecation on the bed. Of the patients with a continuous crescent-shaped reflection pattern (R3), 92.9% had hard stool. R3 was found to be significantly associated with a Mini-Mental State Examination score of ≤10. In most of the patients, US detected a continuation of reflection with acoustic shadow in rectal patterns, indicating fecal retention in the rectum. Point-of-care US can be used by nurses to visualize rectal fecal retention as constipation patterns in the older people with physical and cognitive impairment at long-term care facilities.
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http://dx.doi.org/10.3390/healthcare6020055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023545PMC
May 2018

Extracellular loop structures in silkworm ABCC transporters determine their specificities for Cry toxins.

J Biol Chem 2018 06 17;293(22):8569-8577. Epub 2018 Apr 17.

From the Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan and

Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm () ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.
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http://dx.doi.org/10.1074/jbc.RA118.001761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986200PMC
June 2018

Unexpected Replication Boost by Simeprevir for Simeprevir-Resistant Variants in Genotype 1a Hepatitis C Virus.

Antimicrob Agents Chemother 2018 07 26;62(7). Epub 2018 Jun 26.

Department of Gastroenterology, Kanazawa University Hospital, Kanazawa, Ishikawa, Japan.

Simeprevir is a novel NS3/4A protease inhibitor (PI) of hepatitis C virus (HCV). The baseline polymorphism NS3-Q80K is frequently observed in genotype (GT) 1a HCV and often associated with treatment failure in simeprevir-containing regimens. We aimed to elucidate mechanisms of treatment failure due to NS3-Q80K. We included a Q80R mutation in our study and generated a series of Huh-7.5 cell lines, each of which harbored either wild-type GT 1a strain H77S.3 or the Q80K or Q80R variant. The cells were cultured with increasing concentrations of simeprevir, and NS3 domain sequences were determined. The mutations identified by sequence analyses were subsequently introduced into H77S.3. The sensitivity of each mutant to the NS3/4A PIs simeprevir, asunaprevir, grazoprevir, and paritaprevir was analyzed. We introduced the mutations into GT 1b strain N.2 and compared the sensitivity to simeprevir with that of GT 1a strain H77S.3. While simeprevir treatment selected mutations at residue D168, such as D168A/V in the wild-type virus, an additional mutation at residue R155, R155K, was selected in Q80K/R variants at simeprevir concentrations of <2.5 μM. Sensitivity analyses showed that simeprevir concentrations of <1 μM significantly boosted the replication of Q80K/R R155K variants. Interestingly, this boost was not observed with the other NS3/4A PIs or in Q80R R155Q/G/T/W variants or GT 1b isolates. The boosted replication of the Q80K+R155K variant by simeprevir could be related to treatment failure in simeprevir-containing antiviral treatments in GT 1a HCV-infected patients with the NS3-Q80K polymorphism. This result provides new insight into how resistance-associated variants can cause treatment failure.
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http://dx.doi.org/10.1128/AAC.02601-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021633PMC
July 2018

Assessment of rectal feces storage condition by a point-of-care pocket-size ultrasound device for healthy adult subjects: A preliminary study.

Drug Discov Ther 2018;12(1):42-46

Global Nursing Research Center, Graduate School of Medicine, The University of Tokyo.

The aim of this study was to assess rectal feces storage condition by a pocket-size ultrasonography (PUS) in healthy adults so as to define normal rectal defecation desire. Participants were first assessed rectum by PUS imaging immediately after defecation desire (pre-defecation). Nurses checked the amount and quality of the participants' feces using King's Stool Chart and Bristol stool scale. Finally, PUS was performed for defecation with no defecation desire (post-defecation). Pre-defecation PUS detected high echo area in all patients. All of the post-defecation PUS did not detect high echo area (perfectly no recognizable high echo area in 54.5%, high echo line in 36.4%, and low echo of entire circumference in 9.1% of the patients). Average diameter of rectal crescent was 4.22 ± 0.8 cm. Bristol Stool Scale 1 or 2 (indicating hard stool) of pre-defecation PUS indicated high echo area and acoustic shadow in 100% of the patients. This study showed that healthy adult with defecation desire had high average rectal echo area of 4.0 cm in diameter. PUS may be able to define the rectum diameter for defecation desire of elderly people. PUS is capable of assessing fecal retention of the rectum for point-of-care examinations in home care.
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http://dx.doi.org/10.5582/ddt.2018.01001DOI Listing
August 2019

Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes.

Front Mol Neurosci 2017 7;10:408. Epub 2017 Dec 7.

Laboratory of Pharmacology, Osaka University of Pharmaceutical Sciences, Osaka, Japan.

Inwardly rectifying potassium (Kir) 4.1 channels in astrocytes regulate neuronal excitability by mediating spatial potassium buffering. Although dysfunction of astrocytic Kir4.1 channels is implicated in the development of epileptic seizures, the functional mechanisms of Kir4.1 channels in modulating epileptogenesis remain unknown. We herein evaluated the effects of Kir4.1 inhibition (blockade and knockdown) on expression of brain-derived neurotrophic factor (BDNF), a key modulator of epileptogenesis, in the primary cultures of mouse astrocytes. For blockade of Kir4.1 channels, we tested several antidepressant agents which reportedly bound to and blocked Kir4.1 channels in a subunit-specific manner. Treatment of astrocytes with fluoxetine enhanced BDNF mRNA expression in a concentration-dependent manner and increased the BDNF protein level. Other antidepressants (e.g., sertraline and imipramine) also increased the expression of BDNF mRNA with relative potencies similar to those for inhibition of Kir4.1 channels. In addition, suppression of Kir4.1 expression by the transfection of small interfering RNA (siRNA) targeting Kir4.1 significantly increased the mRNA and protein levels of BDNF. The BDNF induction by Kir4.1 siRNA transfection was suppressed by the MEK1/2 inhibitor U0126, but not by the p38 MAPK inhibitor SB202190 or the JNK inhibitor SP600125. The present results demonstrated that inhibition of Kir4.1 channels facilitates BDNF expression in astrocytes primarily by activating the Ras/Raf/MEK/ERK pathway, which may be linked to the development of epilepsy and other neuropsychiatric disorders.
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http://dx.doi.org/10.3389/fnmol.2017.00408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768989PMC
December 2017

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin.

Insect Biochem Mol Biol 2017 12 8;91:44-54. Epub 2017 Nov 8.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka 2-24-16, Koganei, Tokyo 184-8588, Japan. Electronic address:

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that DYWL of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around DYWL of ECL 4 in the ABCC2.
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http://dx.doi.org/10.1016/j.ibmb.2017.11.002DOI Listing
December 2017

Oral pemphigus vulgaris: Liquid-based cytological findings and pitfalls.

Diagn Cytopathol 2018 Jan 11;46(1):63-66. Epub 2017 Aug 11.

Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.

Pemphigus vulgaris (PV) is a chronic autoimmune bullous disease characterized by the formation of suprabasal cleavage and acantholysis. As this disease almost always affects the oral mucosa, conventional cytological smears of oral lesions can be used for the initial diagnosis of PV. We report two cases of PV that were initially diagnosed based on cytological smears of an oral sample. As atypical squamous cells were present even in the liquid-based cytological (LBC) smears of the oral lesion in these two cases, this ultimately led to the misinterpretation of squamous cell carcinoma. These findings demonstrate that cytological mimicry of oral PV can occur in malignant cases when there is an absence of appropriate clinical information.
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http://dx.doi.org/10.1002/dc.23792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763399PMC
January 2018

Cry toxin specificities of insect ABCC transporters closely related to lepidopteran ABCC2 transporters.

Peptides 2017 Dec 14;98:86-92. Epub 2017 Apr 14.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. Electronic address:

In this study, we examined insect and human ABCC transporters closely related to the lepidopteran ABC transporter C2 (ABCC2), a powerful receptor for the Bacillus thuringiensis Cry toxin, for their responses to various Cry toxins. ABCC2 and the lepidopteran ABC transporter C3 (ABCC3) conferred cultured cells with susceptibility to a lepidopteran-specific Cry1Aa toxin but not to lepidopteran-specific Cry1Ca and Cry1Da. One coleopteran ABCC transporter specifically responded to a coleopteran-specific Cry8Ca toxin. ABCC transporters from a dipteran insect and humans did not respond to any of the tested Cry toxins that are active to lepidopteran and coleopteran insects. These results yield important information for our understanding of insect specificity of Cry toxins and provide the first demonstration of a coleopteran ABCC transporter that serves as a Cry toxin receptor.
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http://dx.doi.org/10.1016/j.peptides.2017.04.003DOI Listing
December 2017

Functional characterization of Bacillus thuringiensis Cry toxin receptors explains resistance in insects.

FEBS J 2016 12 29;283(24):4474-4490. Epub 2016 Nov 29.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.

Bacillus thuringiensis produces Cry toxins, which are used as insecticides in sprays and in transgenic crops. However, little is known about the function of Cry toxin receptors and the mechanisms that determine their binding specificity and activity. In this study, the cRNAs of Bombyx mori ABC transporter C2 (BmABCC2), the toxin-binding region of cadherin-like receptor (BtR175-TBR), or aminopeptidase N1 (BmAPN1) were injected into Xenopus oocytes, and the Cry1Aa-dependent cation-selective pore formation activities of these receptors were analyzed using a two-electrode voltage clamp. Cation current passing through the pores was detected within 25 s, and increased in a linear fashion in BmABCC2-expressing oocytes treated with 88 nm Cry1Aa. This result suggested that Cry1Aa continuously made stable pores with the help of BmABCC2. In contrast, no cation current was observed until 60 min after incubation with 500 nm Cry1Aa in BtR175TBR-expressing oocytes even though oligomerization of Cry1Aa progressed. This result indicated that in the presence of BtR175-TBR most of the oligomerized toxin could not enter the cell membrane. However, oocytes that simultaneously expressed both receptors demonstrated that BtR175-TBR exerted a synergistic effect with BmABCC2 on pore formation in the presence of 22 nm Cry1Aa. These results confirm that the main reason for moderate-level resistance in insects lacking the cadherin-like receptor but expressing ABCC2 is the absence of a similar synergistic promotion of toxin oligomerization. Similar to results from our previous report evaluating ectopic expression in the Sf9/Baculovirus system, BmAPN1 could not by itself cause Cry1A-related pore formation, despite the fact that BmAPN1 gathered toxin on the oocytes as well as BmABCC2 did.
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http://dx.doi.org/10.1111/febs.13952DOI Listing
December 2016

Prospective, randomized, evaluator-blinded study of the long pulse 532-nm KTP laser alone or in combination with the long pulse 1064-nm Nd: YAG laser on facial rejuvenation in Asian skin.

Lasers Surg Med 2016 11 8;48(9):844-851. Epub 2016 Sep 8.

Aoyama Institute of Women's Medicine, Tokyo Women's Medical University, Tokyo 107-0061, Japan.

Background And Objective: Hyperpigmentation is a common concern in Asian patients. Few published studies address overall skin rejuvenation in this group using long-pulse (LP) laser to target pigmentation and stimulate dermal remodeling. The LP KTP 532-nm laser (LP 532-nm) is used primarily to remove epidermal lesions, while the LP Nd: YAG 1064-nm laser (LP 1064-nm) is used to stimulate dermal remodeling in Asian patients with varying efficacy. The LP 532-nm used alone and in combination with LP 1064-nm to reduce pigmentation and rejuvenate skin was previously evaluated in lighter skin, but not in Asian skin. We evaluated the safety and effectiveness of using LP 532-nm for overall photorejuvenation, with and without LP 1064-nm.

Study Design/materials And Methods: Four treatments were administered at 3-week intervals to 22 Japanese females with photodamaged facial skin and bilateral solar lentigines. A direct split-face treatment with LP 532-nm was used on the full-face, and an additional, randomized LP 1064 treatment was administered to one-half of the face. Patients were not fully aware which side of the face was treated with which treatment. Results were evaluated at each treatment, and at 1- and 3-month follow-up visits.

Results: Scoring of a modified pigment severity index (mPSI) and measurement of the melanin index (MI) showed that facial skin treated with LP 532-nm alone and in combination with LP 1064-nm resulted in improvement at the 1- and 3-month follow-up (P < 0.001), but there was no difference between the two sides of the face. Notably, the three dimensional analysis of skin surface showed improvements for the dual-wavelength treatments with significant differences between the two sides (P = 0.003). Most patients reported moderate improvement and were extremely satisfied or satisfied with the outcome. Adverse events were minor and rare.

Conclusions: Pigment-related skin rejuvenation using LP 532-nm appears to be safe and effective for Asian skin. The addition of LP 1064-nm showed no clinical difference but the subtle difference was detected by the 3D analyzing device. Lasers Surg. Med. 48:844-851, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/lsm.22582DOI Listing
November 2016

Expression of the fructose receptor BmGr9 and its involvement in the promotion of feeding, suggested by its co-expression with neuropeptide F1 in Bombyx mori.

Insect Biochem Mol Biol 2016 08 7;75:58-69. Epub 2016 Jun 7.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei 2-24-16, Tokyo 184-8588, Japan. Electronic address:

Insect gustatory receptors (Grs) are members of a large family of proteins with seven transmembrane domains that provide insects with the ability to detect chemical signals critical for feeding, mating, and oviposition. To date, 69 Bombyx mori Grs (BmGrs) genes have been identified via genome studies. BmGr9 has been shown to respond specifically to fructose and to function as a ligand-gated ion channel selectively activated by fructose. However, the sites where this Gr are expressed remain unclear. We demonstrated using reverse transcription (RT)-PCR that BmGr9 is widely expressed in the central nervous system (CNS), as well as oral sensory organs. Additionally, immunohistochemistry was performed using anti-BmGr9 antiserum to show that BmGr9 is expressed in cells of the oral sensory organs, including the maxillary galea, maxillary palps, labrum, and labium, as well as in putative neurosecretory cells of the CNS. Furthermore, double immunohistochemical analysis showed that most BmGr9-expressing cells co-localized with putative neuropeptide F1-expressing cells in the brain, suggesting that BmGr9 is involved in the promotion of feeding behaviors. In addition, a portion of BmGr9-expressing cells in the brain co-localized with cells expressing BmGr6, a molecule of the sugar receptor clade, suggesting that sugars other than fructose are involved in the regulation of feeding behaviors in B. mori larvae.
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http://dx.doi.org/10.1016/j.ibmb.2016.06.001DOI Listing
August 2016

Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins.

Peptides 2016 Apr 27;78:99-108. Epub 2016 Feb 27.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan. Electronic address:

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.
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http://dx.doi.org/10.1016/j.peptides.2016.01.006DOI Listing
April 2016

Mechanisms of nodule-specific melanization in the hemocoel of the silkworm, Bombyx mori.

Insect Biochem Mol Biol 2016 Mar 18;70:10-23. Epub 2015 Dec 18.

Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic address:

In the insect immune system, nodules are known to be a product of the cellular response against microorganisms and may be a preferential target for melanization. However, the mechanism of nodule-preferential melanization remains to be explored. In this study, we identified several mechanisms of nodule-preferential melanization by analyzing congregation and the activation of several factors involved in the prophenoloxidase (proPO)-activating system in the silkworm, Bombyx mori. Microorganism-binding assays revealed that B. mori larval plasma have an effective invading microorganism-surveillance network consisting of at least six pattern-recognition receptors (PRRs). We also found that a hemolymph serine proteinase, BmHP14, can bind to Saccharomyces cerevisiae. Pull-down assays showed that PRR C-type lectins form protein complexes with serine proteinase homologs, BmSPH1 and BmSPH2, which leads to the activated forms of BmSPH1 and BmSPH2 being gathered on microorganisms and trapped in nodules. Immunostaining analysis revealed that most factors in the proPO-activating system and some factors in the triggering system for antimicrobial peptide production exist in the granules of hemocytes which can gather in nodules. Western blot analysis showed that factors in the proPO-activating system are congregated in formed nodules by their concentration in plasma and aggregating hemocytes.
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http://dx.doi.org/10.1016/j.ibmb.2015.12.005DOI Listing
March 2016

Effect of the Nature of Subsequent Environment on Oxytocin and Cortisol Secretion in Maltreated Children.

Front Psychiatry 2015 11;6:173. Epub 2015 Dec 11.

Division of Developmental Higher Brain Functions, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui , Fukui , Japan ; Research Center for Child Mental Development, University of Fukui , Fukui , Japan.

Childhood maltreatment (CM), including abuse and neglect, is a crucial factor that distorts child development. CM is associated with alterations in numerous brain regions, and may be associated with hormonal dysregulation. This study aimed to investigate differences in secretion patterns of cortisol (CT) and oxytocin (OT) among children who experienced CM, children living in residential care facilities and in unstable environments. Among 38 maltreated children, 23 (mean age = 12.2 years, SD = 3.0) were categorized as "Settled" and 15 (mean age = 13.1 years, SD = 2.2) as "Unsettled." Twenty-six age- and gender-matched (mean age = 12.6 years, SD = 2.1), typically developing (TD) children were also included. Clinical and psychological assessments, including IQ and trauma evaluations, were conducted for all participants. Age, gender, and full-scale IQ were used as covariates in hormone analysis. Two saliva samples were collected, one on awakening and the other at bedtime. There were significant differences in the awakening CT levels of the "Unsettled" group, and in bedtime OT levels in the "Settled" group as compared with TD children, and between CM groups. Furthermore, there was a significant difference in trauma-symptomatic depression scores between the "Settled" and "Unsettled" CM group. These results suggest that CT diurnal secretions tend to be reactive to current stress rather than previous experience. OT diurnal secretions are presumably hyper-regulated for coping with the environment to survive and thrive. By measuring salivary CT/OT diurnal patterns, hormonal dysregulation of CM children living in "Settled" environments and "Unsettled" environments was indicated.
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http://dx.doi.org/10.3389/fpsyt.2015.00173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4677106PMC
December 2015

Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

BMC Biol 2015 Jul 8;13:48. Epub 2015 Jul 8.

Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Background: Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery.

Results: Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation.

Conclusions: With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.
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http://dx.doi.org/10.1186/s12915-015-0160-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495774PMC
July 2015

Visual attention for social information and salivary oxytocin levels in preschool children with autism spectrum disorders: an eye-tracking study.

Front Neurosci 2014 17;8:295. Epub 2014 Sep 17.

Research Center for Child Mental Development, University of Fukui Fukui, Japan ; Department of Child Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University, University of Fukui Fukui, Japan.

This study was designed to ascertain the relationship between visual attention for social information and oxytocin (OT) levels in Japanese preschool children with autism spectrum disorder (ASD). We hypothesized that poor visual attention for social information and low OT levels are crucially important risk factors associated with ASD. We measured the pattern of gaze fixation for social information using an eye-tracking system, and salivary OT levels by the Enzyme-Linked Immunosorbent Assay (ELISA). There was a positive association between salivary OT levels and fixation duration for an indicated object area in a finger-pointing movie in typically developing (TD) children. However, no association was found between these variables in children with ASD. Moreover, age decreased an individual's attention to people moving and pointed-at objects, but increased attention for mouth-in-the-face recognition, geometric patterns, and biological motions. Thus, OT levels likely vary during visual attention for social information between TD children and those with ASD. Further, aging in preschool children has considerable effect on visual attention for social information.
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http://dx.doi.org/10.3389/fnins.2014.00295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166357PMC
October 2014

Unexpected ovarian malignancy found after laparoscopic surgery in patients with adnexal masses--a single institutional experience.

Nagoya J Med Sci 2014 Feb;76(1-2):83-90

Laparoscopy has become the standard surgery for the treatment of benign ovarian tumors. The aim of this study was to evaluate the appropriateness of laparoscopy for ovarian tumors, including those with malignant potential. A total of 487 patients with adnexal masses underwent laparoscopic surgery in Social Insurance Chukyo Hospital from January 2000 to December 2012. We reviewed 471 cases that fulfilled the criteria set for this study, and examined 10 cases with unexpected ovarian malignancy to analyze their preoperative diagnosis, second surgery, postoperative chemotherapy, and prognosis. The ages of the 471 patients ranged from 13 to 50 years, with a median of 31. Nulliparous patients numbered 321(68.1%). Of all, 436 patients mostly consisted of those with endometrioma, benign ovarian neoplasm or functional cyst. In all, we histologically identified 10 women with malignancy: 6 with borderline ovarian tumors (BOT), 2 with ovarian cancer, and 2 with histologically rare tumors (immature teratoma and granulosa cell tumor). All patients with BOT were diagnosed with a mucinous histology. Two patients underwent both second radical surgery (hysterectomy and contra- or bilateral salpingo-oophorectomy) and chemotherapies that consisted of CBDCA and PTX or DTX. Thus, 2 patients underwent staging procedures, but the remaining 8 cases did not. None of them had evidence of recurrences. With accurate staging and careful postoperative follow-up, laparoscopic surgery could be a feasible initial operation for patients with adnexal masses including early-stage ovarian malignancy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345716PMC
February 2014

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin-like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins.

Microbiologyopen 2014 Aug 16;3(4):568-77. Epub 2014 Jul 16.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588, Japan.

Directed evolution of a Cry1Aa toxin using phage display and biopanning was performed to generate an increased binding affinity to the Bombyx mori cadherin-like receptor (BtR175). Three mutant toxins (371 WGLA374 , 371 WPHH374 , 371 WRPQ374 25) with 16-, 16-, and 50-fold higher binding affinities, respectively, for BtR175 were selected from a phage library containing toxins with mutations in domain II loop 2. However, the observed toxicities of the three mutants against B. mori larvae and cultured cells expressing the BtR175 toxin-binding region did not increase, suggesting that increased binding affinity to cadherins does not contribute to the insecticidal activity. Affinity maturation of a Cry toxin to a receptor via directed evolution was relatively simple to achieve, and seems to have potential for generating a toxin with increased insecticidal activity.
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http://dx.doi.org/10.1002/mbo3.188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287183PMC
August 2014

Immunohistochemical analysis of the role of hemocytin in nodule formation in the larvae of the silkworm, Bombyx mori.

J Insect Sci 2013 ;13:125

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei 2-24-16, Tokyo 184-8588, Japan.

Hemocytin, a multidomain protein from Bombyx mori L. (Lepidoptera: Bombycidae), is an ortholog of von Willebrand factor and is expected to be a major mediator of hemocyte aggregation. Antiserum was generated against hemocytin, and immune staining of hemocytes, hemolymph, and nodules was performed. Hemocytin was observed in steady-state hemocytes but not in plasma, even after bacterial injection. When hemolymph was smeared on glass slides, hemocytin-containing fibrous structures formed a cellular network mainly consisting of granulocytes and oenocytoids. Hemocytin was stained only in the granules of the granulocytes. When nodule-like aggregates formed 30 sec after bacterial injection, both granulocytes and bacterial cells were observed binding to hemocytin-containing fibrous structures. When nodule sections were stained with antiserum, hemocytin was seen in the matrix of the nodules surrounding the hemocytes. These data suggest that hemocytin plays a major role in nodule formation as a component of the sticky fibrous structure exocytosed from granulocytes.
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http://dx.doi.org/10.1673/031.013.12501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015701PMC
January 2015
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