Publications by authors named "Atsushi Asano"

89 Publications

Functional difference of ATP-generating pathways in rooster sperm (Gallus gallus domesticus).

Anim Reprod Sci 2021 Sep 9;233:106843. Epub 2021 Sep 9.

Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. Electronic address:

Adenosine triphosphate (ATP) production via glycolysis and oxidative phosphorylation is essential for the maintenance of flagellar motility in sperm; however, the primary energy production pathways supporting fertilization vary among species. Inconsistency in thought exists regarding which pathways maintain ATP production and sperm motility in poultry. Glycolysis and mitochondrial oxidation contribute to flagellar motion in chicken sperm, but the relative dependence on these pathways for motility and penetrability into the inner perivitelline layer remains unclear. In the present study, there was use of various inhibitors and energy substrates to evaluate the relative contribution of anaerobic glycolysis and mitochondrial oxidation to chicken sperm flagellar motility, ATP production, and penetrating capacity through the perivitelline layer. Although both pathways contributed to these processes to varying extent, glucose was the primary substrate for sperm penetration into the inner perivitelline layer in chickens. Furthermore, results from metabolic stress analyses indicated that there was less perivitelline penetrability in response to pyruvate that was not due to changes in reactive oxygen species or intracellular pH. Overall, results from the present study indicate glycolysis and mitochondrial oxidation pathways have distinct functions in the flagellar motility and penetrability of the perivitelline membrane by rooster sperm. There, therefore, are new insights as a result of findings in the present study into the energy production system of sperm through which there is utilization of extracellular metabolic substrates for maintaining sperm fertilization capacity.
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http://dx.doi.org/10.1016/j.anireprosci.2021.106843DOI Listing
September 2021

Mechanically Robust, Self-Healable Polymers Usable under High Humidity: Humidity-Tolerant Noncovalent Cross-Linking Strategy.

J Am Chem Soc 2021 Sep 25;143(37):15279-15285. Epub 2021 Aug 25.

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

Although mechanically robust polymer materials had not been thought to self-heal, we recently found that poly(ether thiourea) PTUEG, which is a glassy polymer with high mechanical strength, self-heals even at ambient temperatures. This finding updated the above preconception. Nevertheless, it should also be noted that PTUEG, under high humidity, absorbs water and is plasticized to lose its mechanical strength. Humidity-induced plasticization is a general problem for polymers with polar groups. Herein, we report that PTUEG, if designed by copolymerization to contain only 10 mol % of a dicyclohexylmethane (CyM) thiourea unit (TUCyM), serves as a humidity-tolerant, mechanically robust polymer material that can self-heal at ambient temperatures. This copolymer contained, in its ether thiourea (TUEG)-rich domain, a humidity-tolerant, noncovalently cross-linked 3D network with mechanical robustness formed by stacking of the CyM group. The present work provides a promising design strategy for mechanically robust, self-healable polymers usable under high humidity.
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http://dx.doi.org/10.1021/jacs.1c06494DOI Listing
September 2021

[OUTCOMES OF CHRONIC UNILATERAL HEMATURIA TREATED USING DIGITAL FLEXIBLE URETEROSCOPE].

Nihon Hinyokika Gakkai Zasshi 2020 ;111(1):16-21

Department of Urology, Jinyukai Hospital.

(Objectives) We examined the treatment outcomes in cases of chronic unilateral hematuria treated using flexible ureteroscope for observation and hemostasis. (Methods) The study included 14 patients (7 men and 7 women) with a median age of 56.5 years who underwent ureteroscopy using a digital flexible ureteroscope for chronic unilateral hematuria between March 2014 and August 2019. All the patients presented with macroscopic hematuria as a clinical symptom, but in one patient, the hematuria was accompanied by anemia and required a blood transfusion. In addition, bleeding occurred on the left side in 8 patients and on the right side in 3 patients; however, for the remaining 3 patients, the affected side could not be identified. Fourteen patients were examined on the basis of the ureteroscopic findings, number of bleeding sites, hemostatic intervention, treatment effect, and presence or absence of recurrences. (Results) The ureteroscopic findings showed a hemangioma in 7 patients and minute venous rupture in 3, but the remaining 4 patients showed no clear findings. The site of the findings was in the superior calyces in 8 cases, middle calyces in 4 cases, inferior calyces in 4 cases, and renal pelvic wall in 1 case. In addition, the findings were located at multiple sites in 6 cases, including all renal calyces in 2 cases. Ten patients with findings underwent hemostatic interventions (electrocoagulation and laser treatment). The median postoperative follow-up period was 32.4 months (range, 6.4-65.4 months). In all the cases, the hematuria disappeared after treatment. One of the 2 patients with findings in all renal calyces showed recurrence of macroscopic hematuria at 1 year and 6 months, which disappeared after conservative treatment. (Conclusions) In this study, observation using digital flexible ureteroscope was useful in the treatment of chronic unilateral hematuria, and the hemostatic interventions performed on the bleeding sites in the renal pelvis were effective.
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http://dx.doi.org/10.5980/jpnjurol.111.16DOI Listing
February 2021

Src family kinases-mediated negative regulation of sperm acrosome reaction in chickens (Gallus gallus domesticus).

PLoS One 2020 12;15(11):e0241181. Epub 2020 Nov 12.

Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.

The acrosome reaction (AR) is a strictly-regulated, synchronous exocytosis that is required for sperm to penetrate ova. This all-or-nothing process occurs only once in the sperm lifecycle through a sequence of signaling pathways. Spontaneous, premature AR therefore compromises fertilization potential. Although protein kinase A (PKA) pathways play a central role in AR across species, the signaling network used for AR induction is poorly understood in birds. Mechanistic studies of mammalian sperm AR demonstrate that PKA activity is downstreamly regulated by Src family kinases (SFKs). Using SFK inhibitors, our study shows that in chicken sperm, SFKs play a role in the regulation of PKA activity and spontaneous AR without affecting motility. Furthermore, we examined the nature of SFK phosphorylation using PKA and protein tyrosine phosphatase inhibitors, which demonstrated that unlike in mammals, SFK phosphorylation in birds does not occur downstream of PKA and is primarily regulated by calcium-dependent tyrosine phosphatase activity. Functional characterization of SFKs in chicken sperm showed that SFK activation modulates the membrane potential and plays a role in inhibiting spontaneous AR. Employing biochemical isolation, we also found that membrane rafts are involved in the regulation of SFK phosphorylation. This study demonstrates a unique mechanism for regulating AR induction inherent to avian sperm that ensure fertilization potential despite prolonged storage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241181PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660528PMC
December 2020

Localisation and function of glucose transporter GLUT1 in chicken (Gallus gallus domesticus) spermatozoa: relationship between ATP production pathways and flagellar motility.

Reprod Fertil Dev 2020 Apr;32(7):697-705

Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan; and Corresponding author. Email:

Glucose plays an important role in sperm flagellar motility and fertility via glycolysis and oxidative phosphorylation, although the primary mechanisms for ATP generation vary between species. The glucose transporter 1 (GLUT1) is a high-affinity isoform and a major glucose transporter in mammalian spermatozoa. However, in avian spermatozoa, the glucose metabolic pathways are poorly characterised. This study demonstrates that GLUT1 plays a major role in glucose-mediated motility of chicken spermatozoa. Using specific antibodies and ligand, we found that GLUT1 was specifically localised to the midpiece. Sperm motility analysis showed that glucose supported sperm movement during incubation for 0-80min. However, this was abolished by the addition of a GLUT1 inhibitor, concomitant with a substantial decrease in glucose uptake and ATP production, followed by elevated mitochondrial activity in response to glucose addition. More potent inhibition of ATP production and mitochondrial activity was observed in response to treatment with uncouplers of oxidative phosphorylation. Because mitochondrial inhibition only reduced a subset of sperm movements, we investigated the localisation of the glycolytic pathway and showed glyceraldehyde-3-phosphate dehydrogenase and hexokinase I at the midpiece and principal piece of the flagellum. The results of this study provide new insights into the mechanisms involved in ATP production pathways in avian spermatozoa.
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http://dx.doi.org/10.1071/RD19240DOI Listing
April 2020

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions.

Biopolymers 2020 Jun 23;111(6):e23352. Epub 2020 Mar 23.

Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa, Japan.

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular β-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular β-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.
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http://dx.doi.org/10.1002/bip.23352DOI Listing
June 2020

Compatibility Evaluation of Non-Woven Sheet Composite of Silk Fibroin and Polyurethane in the Wet State.

Polymers (Basel) 2018 Aug 6;10(8). Epub 2018 Aug 6.

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.

SF/polyurethane composite non-woven sheet was fabricated to evaluate the cardiovascular tissue engineering materials in the wet state. The compatibility and microstructure analyses were carried out on the fabricated SF/polyurethane composite non-woven sheet by thermal analysis and solid-state NMR analysis in the wet state. To evaluate the modulus of elasticity, a tensile test was performed and supported with dynamic viscoelasticity and mechanical analysis. Results showed that SF/polyurethane composites form domains within the non-woven sheet and are in a finely dispersed state while maintaining their structures at a scale of several tens of nm. Moreover, an increase of the loss tangent with low elastic modulus proved that a micromolecular interaction occurs between silk fibroin (SF) and polyurethane molecules.
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http://dx.doi.org/10.3390/polym10080874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403721PMC
August 2018

Membrane raft-mediated regulation of glucose signaling pathway leading to acrosome reaction in chicken sperm†.

Biol Reprod 2019 06;100(6):1482-1491

Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki-, Japan.

Despite knowledge that glucose metabolism is essential for the regulation of signaling cascades in the sperm that are pre-assembled into specific areas and function at multistage for fertilization, the physiological roles of glucose in avian sperm are poorly understood. Accumulated results of studies conducted in our laboratory and others indicate that sperm possess membrane microdomains, or membrane rafts, which play important roles in several processes, including the induction of acrosome reaction (AR). When characterizing proteomes associated with chicken sperm rafts, we observed marked enrichment of glucose transporter 3 (GLUT3). Here we show that glucose uptake is mediated by membrane rafts and stimulates AR induction by activating AMP-activated protein kinase (AMPK). Using a specific antibody, we observed that GLUT3 is localized to the entire flagellum and acrosome region and highly associated with membrane rafts. The addition of glucose stimulated AR in a dose-dependent manner without affecting sperm motility. AR and glucose uptake assays were performed using both inhibitors and activators, and demonstrated that glucose-dependent AR results from the activity of a glucose transporter located in membrane rafts and associated with AMPK. To better understand the mechanism of AMPK activation by glucose, we evaluated localization and phosphorylation status of AMPKα, showing that glucose uptake stimulates AMPKα phosphorylation, leading to its complete activation. Together, these results lead us to propose a novel mechanism by which glucose uptake stimulates the AMPK signaling pathway via membrane rafts, resulting in maximal acrosomal responsiveness in avian sperm as migrating upward to a fertilization site.
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http://dx.doi.org/10.1093/biolre/ioz015DOI Listing
June 2019

Angelica keiskei (Ashitaba) powder and its functional compound xanthoangelol prevent heat stress-induced impairment in sperm density and quality in mouse testes.

J Reprod Dev 2019 Apr 26;65(2):139-146. Epub 2019 Jan 26.

Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8577, Japan.

Recently, gradual decline in human sperm production has become a serious worldwide concern because it leads to increased rates of infertility. Endocrine disrupters, lifestyle changes, and varicocele, all of which elevate testicular temperature, are thought to be the main causes of this decline. The present study aimed to determine whether the dietary phytochemicals Angelica keiskei (Ashitaba) powder (57.5 mg/kg) and its functional component, xanthoangelol (3 mg/kg), can prevent heat stress-induced impairment in sperm density and quality in mice. Sperm parameters were analyzed 28 days after mice exposure to heat. Supplementation with Ashitaba powder completely prevented heat-induced impairment in sperm parameters, including densities of motile sperms and progressive sperms (> 25 μm/sec), and amplitude of lateral head displacement. Xanthoangelol did not exert a complete protective effect; nevertheless, it significantly prevented heat stress-induced reduction in most parameters. Both Ashitaba powder and xanthoangelol elevated the expression of the widely expressed heat shock proteins (HSPs) Hspa1a and Hsp40 and the antioxidant enzyme glutathione synthase in non-stressed testes. Ashitaba powder significantly prevented heat stress-induced reduction in the expression of Hspa1l and Hspa2, which are highly expressed in the testes and critical for fertility. Our results showed that Ashitaba powder and xanthoangelol protected testicular cells from heat stress, probably by elevating the levels of antioxidant enzymes and HSPs. Supplementation with dietary functional phytochemicals may help prevent heat stress-induced male infertility.
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http://dx.doi.org/10.1262/jrd.2018-141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6473112PMC
April 2019

Membrane rafts regulate sperm acrosome reaction via cAMP-dependent pathway in chickens (Gallus gallus domesticus).

Biol Reprod 2018 11;99(5):1000-1009

Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.

Both transcriptionally and translationally inactive sperm need preassembled pathways into specific cellular compartments to function. Although initiation of the acrosome reaction (AR) involves several signaling pathways including protein kinase A (PKA) activation, how these are regulated remains poorly understood in avian sperm. Membrane rafts are specific membrane regions enriched in sterols and functional proteins and play important roles in diverse cellular processes, including signal transduction. Our recent studies on chicken sperm demonstrated that membrane rafts exist and play a role in multistage fertilization. These, combined with the functional importance of membrane rafts in mammalian sperm AR, prompted us to investigate the roles of membrane rafts in signaling pathways leading to AR in chicken sperm. Using 2-hydroxypropyl-β-cyclodextrin (2-OHCD), we found that the disruption of membrane rafts inhibits PKA activity and AR without affecting protein tyrosine phosphorylation; however, these inhibitions were abolished in the presence of a cyclic 3,5-adenosine monophosphate (cAMP) analog. In addition, biochemical experiments showed a decrease in cAMP content in 2-OHCD-treated sperm, suggesting the involvement of soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclase (tmAC). Pharmacological experiments, combined with transcriptome analysis, showed that sAC and tmAC are present and involved in AR induction in chicken sperm. Furthermore, stimulation of both isoforms reversed the inhibition of PKA activity and AR in 2-OHCD-treated sperm. In conclusion, our results demonstrated that membrane rafts play an important role in AR induction by regulating the cAMP-dependent pathway and that they provide a mechanistic insight into membrane regulation of AR and sperm function in birds.
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http://dx.doi.org/10.1093/biolre/ioy120DOI Listing
November 2018

Cysteine dioxygenase is essential for mouse sperm osmoadaptation and male fertility.

FEBS J 2018 05 16;285(10):1827-1839. Epub 2018 Apr 16.

The Baker Institute for Animal Health, Cornell University, Ithaca, NY, USA.

Sperm entering the epididymis are immotile and cannot respond to stimuli that will enable them to fertilize. The epididymis is a highly complex organ, with multiple histological zones and cell types that together change the composition and functional abilities of sperm through poorly understood mechanisms. Sperm take up taurine during epididymal transit, which may play antioxidant or osmoregulatory roles. Cysteine dioxygenase (CDO) is a critical enzyme for taurine synthesis. A previous study reported that male CDO mice exhibit idiopathic infertility, prompting us to investigate the functions of CDO in male fertility. Immunoblotting and quantitative reverse transcription-polymerase chain reaction analysis of epididymal segments showed that androgen-dependent CDO expression was highest in the caput epididymidis. CDO mouse sperm demonstrated a severe lack of in vitro fertilization ability. Acrosome exocytosis and tyrosine phosphorylation profiles in response to stimuli were normal, suggesting normal functioning of pathways associated with capacitation. CDO sperm had a slight increase in head abnormalities. Taurine and hypotaurine concentrations in CDO sperm decreased in the epididymal intraluminal fluid and sperm cytosol. We found no evidence of antioxidant protection against lipid peroxidation. However, CDO sperm exhibited severe defects in volume regulation, swelling in response to the relatively hypo-osmotic conditions found in the female reproductive tract. Our findings suggest that epididymal CDO plays a key role in post-testicular sperm maturation, enabling sperm to osmoregulate as they transition from the male to the female reproductive tract, and provide new understanding of the compartmentalized functions of the epididymis.
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http://dx.doi.org/10.1111/febs.14449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992081PMC
May 2018

[A CASE OF BILATERAL SYNCHRONOUS TESTICULAR TUMORS, WHICH WAS DIFFICULT TO DIFFERENTIATE FROM MALIGNANT LYMPHOMA].

Nihon Hinyokika Gakkai Zasshi 2018 ;109(3):160-163

Department of Urology, National Defense Medical College.

We report a case of bilateral synchronous testicular tumors, which was difficult to differentiate from malignant lymphoma. A 56-year-old man with a left scrotal mass was referred to our hospital. Ultrasonography revealed a uniformly hypoechoic mass in bilateral testes. Magnetic resonance imaging also revealed a homogeneously low-intensity lesion in the bilateral testes on T2-weighted images. Abdominal and chest computed tomography showed no lymphadenopathy or metastasis. The image findings at that time suggested a malignant lymphoma, and consequently, we performed a right radical orchiectomy. Histopathological examination revealed typical seminoma in the right testis; following this observation, left radical orchiectomy was performed, and the patient was diagnosed with synchronous bilateral testicular germ cell tumors. No recurrence or metastasis has been detected postoperatively. We recommend that the diagnosis of bilateral testicular tumors be made on the basis of patients' age, tumor marker level, and image findings.
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http://dx.doi.org/10.5980/jpnjurol.109.160DOI Listing
January 2018

Characterization of the functions and proteomes associated with membrane rafts in chicken sperm.

PLoS One 2017 2;12(11):e0186482. Epub 2017 Nov 2.

Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan.

Cellular membranes are heterogeneous, and this has a great impact on cellular function. Despite the central role of membrane functions in multiple cellular processes in sperm, their molecular mechanisms are poorly understood. Membrane rafts are specific membrane domains enriched in cholesterol, ganglioside GM1, and functional proteins, and they are involved in the regulation of a variety of cellular functions. Studies of the functional characterization of membrane rafts in mammalian sperm have demonstrated roles in sperm-egg binding and the acrosomal reaction. Recently, our biochemical and cell biological studies showed that membrane rafts are present and might play functional roles in chicken sperm. In this study, we isolated membrane rafts from chicken sperm as a detergent-resistant membranes (DRM) floating on a density gradient in the presence of 1% Triton X-100, and characterized the function and proteomes associated with these domains. Biochemical comparison of the DRM between fresh and cryopreserved sperm demonstrated that cryopreservation induces cholesterol loss specifically from membrane rafts, indicating the functional connection with reduced post-thaw fertility in chicken sperm. Furthermore, using an avidin-biotin system, we found that sperm DRM is highly enriched in a 60 KDa single protein able to bind to the inner perivitelline layer. To identify possible roles of membrane rafts, quantitative proteomics, combined with a stable isotope dimethyl labeling approach, identified 82 proteins exclusively or relatively more associated with membrane rafts. Our results demonstrate the functional distinctions between membrane domains and provide compelling evidence that membrane rafts are involved in various cellular pathways inherent to chicken sperm.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186482PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667776PMC
December 2017

Development and Preservation of Avian Sperm.

Adv Exp Med Biol 2017 ;1001:59-73

Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8572, Japan.

Terminally differentiated avian sperm consist of a head which male genetic material locates and flagellum that provides the motive force to propel them towards the fertilization site. The apical end of the sperm head accommodates a secretory vesicle, called an acrosome, that undergoes acrosome reaction releasing proteolytic content to penetrate the peri-vitelline membrane of an egg. Transcriptionally and translationally inactive, sperm need to rely on these distinct compartments in which different functions are preassembled, in order to achieve the goal of "fertilization". How are these complex structures with high functionality formed? Spermatogenesis is divided into an early stage in which diploid spermatogonia is proliferated into round spermatids thorough mitotic and meiotic divisions, and a late stage in which round spermatids are transformed into sperm though nuclear condensation and elongation of the sperm head, and formation of accessory structures. Recently, it was reported in aves that morphologically differentiated sperm undergo post-testicular maturation during passage through the male genital tract, suggesting that a similar system to mammals might be involved in the acquisition of fertilizing ability in avian sperm. Investigation for mechanisms underlying how sperm regulate their functions which are necessary to achieve fertilization is important for developing reproductive biotechnology in aves, because cryopreservation of poultry sperm is still not reliable for use in commercial production or for the preservation of genetic resources. In this review, we firstly provide an update on avian spermatogenesis, and then discuss the uniqueness of structure and functions of avian sperm, highlighting differences from mammalian sperm. Lastly, we discuss the molecular mechanism and current techniques of cryopreservation for avian sperm.
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http://dx.doi.org/10.1007/978-981-10-3975-1_4DOI Listing
April 2018

Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice.

Biochem Biophys Rep 2017 Mar 26;9:322-329. Epub 2017 Jan 26.

Laboratory of Veterinary Physiology, Department of Veterinary Medicine, and Laboratory of Animal Therapeutics, Japan.

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing , a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of in mice ( transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from -overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.
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http://dx.doi.org/10.1016/j.bbrep.2017.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614627PMC
March 2017

Modification of membrane cholesterol and desmosterol in chicken spermatozoa improves post-thaw survival and prevents impairment of sperm function after cryopreservation.

Reprod Fertil Dev 2018 Mar;30(4):591-599

Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8577, Japan.

During cryopreservation, spermatozoa are subjected to cryodamage that leads to a decline in fertilisation ability. Due to the complex nature of this process, the initial trigger for cryodamage remains unknown. Recently, we demonstrated that cryopreservation induces early apoptotic changes characterised by phosphatidylserine (PS) translocation via sterol loss from the plasma membrane of chicken spermatozoa. This led us to hypothesise that sterol incorporation into membranes minimises cryodamage, thereby improving the quality of cryopreserved chicken spermatozoa. In the present study, treating spermatozoa with 1.5mgmL-1 cholesterol- and 3mgmL-1 desmosterol-loaded cyclodextrin (CLC and DLC respectively) increased post-thaw survival and motility. These effects appeared to be highly dependent the amount of sterol loaded into the spermatozoa. Localisation experiments confirmed the incorporation of exogenous cholesterol into the sperm head region. Detection of PS translocation showed that elevation of these sterols inhibited early apoptotic changes, thereby enhancing post-thaw survival. Furthermore, CLC and DLC treatment suppressed spontaneous acrosome reaction after cryopreservation, preserving the ability of spermatozoa to undergo acrosome reactions in response to physiological stimulation. These results demonstrate that loading sterols into chicken spermatozoa before cryopreservation enhances their quality by inhibiting early apoptotic changes and spontaneous acrosome reactions. The present study provides new mechanistic insight into cryodamage in chicken spermatozoa.
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http://dx.doi.org/10.1071/RD17076DOI Listing
March 2018

Fluorescent organic single crystals with elastic bending flexibility: 1,4-bis(thien-2-yl)-2,3,5,6-tetrafluorobenzene derivatives.

Sci Rep 2017 08 25;7(1):9453. Epub 2017 Aug 25.

Department of Applied Chemistry, National Defence Academy, 1-10-20 Hashirimizu, Yokosuka, 239-8686, Japan.

Organic single crystals with elastic bending flexibility are rare because they are generally brittle. We report here fluorescent organic single crystals based on thiophene-tetrafluorobenzene-thiophene derivatives, mainly 1,4-bis(thien-2-yl)-2,3,5,6-tetrafluorobenzene. Three derivatives were synthesized by Pd-catalyzed cross-coupling reactions (Stille or direct arylation pathways). The crystallization of the derivatives gave large (mm- or cm-scale) crystals. Two crystals of 1,4-bis(thien-2-yl)-2,3,5,6-tetrafluorobenzene, 1, and 1,4-bis(4-methylthien-2-yl)-2,3,5,6-tetrafluorobenzene, 3, bent under applied stress and quickly recovered its original shape upon relaxation. The other crystal of 1,4-bis(5-methylthien-2-yl)-2,3,5,6-tetrafluorobenzene, 2, showed brittle breakage under applied stress (normal behavior). Fibril lamella crystal structure based on criss-cross packed slip-stacked molecular wires and its structural integrity are important factors for the design and production of next generation crystal materials with elastic bending flexibility. Furthermore, mechanical bending-relaxation resulted in reversible change of the morphology and fluorescence (mechanofluorochromism). Such bendable crystals would lead to the next generation solid-state fluorescent and/or semiconducting materials.
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http://dx.doi.org/10.1038/s41598-017-09848-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573333PMC
August 2017

Comparison of Membrane Characteristics between Freshly Ejaculated and Cryopreserved Sperm in the Chicken.

J Poult Sci 2016 Oct;53(4):305-312

Faculty of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan.

Cryopreserved sperm undergoes serious damage which affects its fertilizing ability. Despite progress in understanding the nature of functional deterioration in mammalian sperm, little is known about the mechanism involved in the induction of functional damage in avian sperm. Cellular membranes are considered the primary site of cryodamage to sperm. Membrane rafts are specific membrane regions enriched in sterols, ganglioside G, and functional proteins and they play important roles in the regulation of diverse functions exerted in mammalian sperm during fertilization. Several reports investigating cryopreservation-induced membrane changes in mammalian sperm have suggested that cryopreservation induces a compositional alteration of membrane rafts via a loss of membrane sterols, leading to impaired fertilizing ability. Recently, we demonstrated that membrane rafts are present in chicken sperm. Therefore, we investigated a possible mechanism for the induction of functional damage in cryopreserved chicken sperm, with particular attention to cryopreservation-induced compositional changes in membrane rafts. Sterol quantification showed that loss of sterols from sperm membranes occurred following cryopreservation. Biochemical analyses of detergent-insoluble membranes showed that the lipid and protein compositions of membrane rafts were altered dramatically by cryopreservation. To determine the physiological role of these changes, we examined external translocation of phosphatidylserine (PS), representing an early apoptotic change, and found that cryopreservation induced apoptotic changes in chicken sperm. Furthermore, methyl--cyclodextrin-induced loss of sterols from the plasma membranes stimulated PS translocation that was not accompanied with caspase-3 activation, which plays an important role downstream of the apoptotic cascade. Based on the results obtained in this study, we discuss a new mechanism for reduction of the fertilizing ability in avian sperm after cryopreservation.
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http://dx.doi.org/10.2141/jpsa.0160043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477166PMC
October 2016

Organization of Membrane Rafts in Chicken Sperm.

J Poult Sci 2016 Jul;53(3):233-239

Faculty of Life and Environmental Sciences, University of Tsukuba, Japan.

Being transcriptionally and translationally inactive, sperm must utilize preassembled pathways into specific compartments in which they function to fertilize ovum. Membrane rafts are specific membrane regions enriched in sterols and glycosphingolipids such as ganglioside G (G) and play an important role in a variety of cellular functions. Recent findings have demonstrated that membrane rafts are present in mammalian sperm and are involved in regulating the induction of acrosome exocytosis. However, no information is available on whether avian sperm possess membrane rafts. Thus, we investigated the organization of membrane rafts in chicken sperm. Our localization experiments for G and sterols showed that the plasma membrane overlaying the sperm head possesses specific membrane domains enriched in both aforementioned lipids. Caveolin-1, which localizes into membrane rafts in other systems, was localized only to the sperm tail. Based on the biochemical definition that membrane rafts are insoluble membranes when subjected to a Triton X-100 treatment, we isolated detergent-insoluble membranes from chicken sperm and quantified the G content, which showed an enrichment of G in the membrane fraction relative to the detergent-soluble fraction. Together with the results of localization and biochemical experiments, we demonstrate for the first time that membrane rafts exist in chicken sperm. Thus, our results provide a foundation for investigating a novel cellular pathway inherent in avian sperm membranes that might be involved in functions necessary to achieve fertilization.
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http://dx.doi.org/10.2141/jpsa.0150160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477140PMC
July 2016

Expression and intracellular localization of TBC1D9, a Rab GTPase-accelerating protein, in mouse testes.

Exp Anim 2015 29;64(4):415-24. Epub 2015 Jun 29.

Laboratory of Veterinary Biochemistry, Faculty of Agriculture, Tottori University, Japan.

Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.
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http://dx.doi.org/10.1538/expanim.15-0016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637379PMC
June 2016

Full-length transient receptor potential vanilloid 1 channels mediate calcium signals and possibly contribute to osmoreception in vasopressin neurones in the rat supraoptic nucleus.

Cell Calcium 2015 Jan 15;57(1):25-37. Epub 2014 Nov 15.

Laboratory of Veterinary Physiology, Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori, Japan; The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan. Electronic address:

Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules.
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http://dx.doi.org/10.1016/j.ceca.2014.11.003DOI Listing
January 2015

Migration and differentiation of gonadal germ cells under cross-sex germline chimeras condition in domestic chickens.

J Reprod Dev 2014 16;60(6):406-10. Epub 2014 Aug 16.

Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan; Fellow of the Japanese Society for the Promotion of Science.

A series of experiments was conducted to investigate migration, proliferation and differentiation of gonadal germ cells (GGCs) collected from the gonads of 7-day-old chick embryos under cross-sex germline chimera conditions. The migratory and proliferative abilities of exogenous GGCs were examined by transferring 50 fluorescently labeled GGCs collected from White Leghorn (WL) embryos into the blood of 2-day-old Rhode Island Red (RIR) embryos. No significant difference was observed in the number of fluorescently labeled GGCs in the gonads of recipient embryos among any of the four possible donor and recipient sex combinations. Cross-sex germline chimeras were produced to examine the differentiation of GGCs by transferring 100 GGCs from WL embryos into 2-day-old RIR embryos. Exogenous-GGC-derived progeny were obtained from both male and female recipients, except when female GGCs were transferred into male recipients. The migratory ability of GGCs recovered from the 7-day-old embryonic gonad was not influenced by cross-sex germ cell transfer conditions, whereas the differentiation of the GGCs was affected by the sex combinations of GGCs donors and recipients.
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http://dx.doi.org/10.1262/jrd.2013-108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284313PMC
October 2015

Fabrication of enzyme-degradable and size-controlled protein nanowires using single particle nano-fabrication technique.

Nat Commun 2014 Apr 28;5:3718. Epub 2014 Apr 28.

Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan.

Protein nanowires exhibiting specific biological activities hold promise for interacting with living cells and controlling and predicting biological responses such as apoptosis, endocytosis and cell adhesion. Here we report the result of the interaction of a single high-energy charged particle with protein molecules, giving size-controlled protein nanowires with an ultra-high aspect ratio of over 1,000. Degradation of the human serum albumin nanowires was examined using trypsin. The biotinylated human serum albumin nanowires bound avidin, demonstrating the high affinity of the nanowires. Human serum albumin-avidin hybrid nanowires were also fabricated from a solid state mixture and exhibited good mechanical strength in phosphate-buffered saline. The biotinylated human serum albumin nanowires can be transformed into nanowires exhibiting a biological function such as avidin-biotinyl interactions and peroxidase activity. The present technique is a versatile platform for functionalizing the surface of any protein molecule with an extremely large surface area.
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http://dx.doi.org/10.1038/ncomms4718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015332PMC
April 2014

Membrane rafts regulate phospholipase B activation in murine sperm.

Commun Integr Biol 2013 Nov 17;6(6):e27362. Epub 2013 Dec 17.

The Baker Institute for Animal Health; College of Veterinary Medicine; Cornell University; Ithaca, NY USA.

It is intuitive that fertilization-the start of life-involves communication between a sperm cell and an egg. It has been known that to become able to fertilize an egg, a sperm must first communicate with stimuli in the female tract. For example, sterol removal from the plasma membrane is required for sperm to undergo membrane fusion during acrosome exocytosis (AE). However, how membrane lipid changes were transduced into initiation of AE remained unclear. Recently, we found that sperm phospholipase B (PLB) is activated in response to sterol removal and released into the extracellular fluid by proteolytic cleavage. The resultant active PLB fragment can stimulate initiation of AE without other physiological stimulation. These results provide a possible mechanism for how AE is triggered, a critical question given recent data from others that show that AE is induced prior to contact with the egg's extracellular covering, the zona pellucida.
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http://dx.doi.org/10.4161/cib.27362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3984294PMC
November 2013

Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

Dev Cell 2014 Feb;28(3):310-21

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Road, Ithaca, NY 14853, USA. Electronic address:

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.
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http://dx.doi.org/10.1016/j.devcel.2014.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947087PMC
February 2014

Demonstration of the clathrin- and caveolin-mediated endocytosis at the maternal-fetal barrier in mouse placenta after intravenous administration of gold nanoparticles.

J Vet Med Sci 2014 Mar 20;76(3):377-87. Epub 2013 Nov 20.

Department of Veterinary Pathology, Tottori University, 4-101 Koyama Minami, Tottori 680-8553, Japan.

Exposure to nanoparticles during pregnancy is a public concern, because nanoparticles may pass from the mother to the fetus across the placenta. The purpose of this study was to determine the possible translocation pathway of gold nanoparticles across the maternal-fetal barrier as well as the toxicity of intravenously administered gold nanoparticles to the placenta and fetus. Pregnant ICR mice were intravenously injected with 0.01% of 20- and 50-nm gold nanoparticle solutions on the 16th and 17th days of gestation. There was no sign of toxic damage to the placentas as well as maternal and fetal organs of the mice treated with 20- and 50-nm gold nanoparticles. ICP-MS analysis demonstrated significant amounts of gold deposited in the maternal livers and placentas, but no detectable level of gold in the fetal organs. However, electron microscopy demonstrated an increase of endocytic vesicles in the cytoplasm of syncytiotrophoblasts and fetal endothelial cells in the maternal-fetal barrier of mice treated with gold nanoparticles. Clathrin immunohistochemistry and immunoblotting showed increased immunoreactivity of clathrin protein in the placental tissues of mice treated with 20- and 50-nm gold nanoparticles; clathrin immunopositivity was observed in syncytiotrophoblasts and fetal endothelial cells. In contrast, caveolin-1 immunopositivity was observed exclusively in the fetal endothelium. These findings suggested that intravenous administration of gold nanoparticles may upregulate clathrin- and caveolin-mediated endocytosis at the maternal-fetal barrier in mouse placenta.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013364PMC
http://dx.doi.org/10.1292/jvms.13-0512DOI Listing
March 2014

Temperature distribution in a solid state NMR sample rotor during MAS experiments.

Anal Sci 2013 ;29(11):1089-93

Department of Applied Chemistry, National Defense Academy.

We investigated temperature distribution in a solid state nuclear magnetic resonance (NMR) sample rotor under magic angle spinning (MAS) experiments by analyzing the (207)Pb chemical shift of solid lead nitrate (Pb(NO3)2). The analysis corrected a mismatch of the temperature dependence of (1)H spin-lattice relaxation time (T1(H)) obtained from different sample positions in the rotor. We prepared three rotors that consisted of Pb(NO3)2 partitioned into the three different parts with Teflon spacers, and measured the temperature distribution related to the placement using the prepared three rotors. The obtained temperature distribution showed a large gradient in the sample rotor. The temperature distribution was related to the placement in the rotor and successfully visualized. Finally, the divergent temperature dependence of T1(H) obtained at different placement positions in the sample rotor was successfully corrected to accurate temperature dependence.
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http://dx.doi.org/10.2116/analsci.29.1089DOI Listing
September 2014

Antiviral and antiproliferative effects of canine interferon-λ1.

Vet Immunol Immunopathol 2013 Nov 27;156(1-2):141-6. Epub 2013 Sep 27.

Laboratory of Veterinary Biochemistry, Tottori University, Tottori 680-8553, Japan.

Interferon (IFN)-λs, members of the type III IFN group, were recently identified in several vertebrates. Although IFN-λs have the potential to be utilized as antiviral and antitumor agents in veterinary medicine, the biological properties of IFN-λs have not yet been studied in companion animals. In this study, we analyzed the expression of canine IFN-λs and their receptors, produced a recombinant canine IFN-λ1 protein, and investigated its antiviral and antiproliferative activities using a canine kidney epithelial cell line, MDCK cells. MDCK cells were found to express type III IFN molecules, IFN-λ1 and IFN-λ3, and the receptors, IFNλR1 and IL10R2. IFN-λ1 was induced faster than IFN-λ3 by stimulation with poly (I:C). His-tagged IFN-λ1 protein expressed in Escherichia coli inhibited cytolytic plaque formation by influenza A virus infection, and induced the expression of interferon-stimulated genes, Mx1 and OAS1, in MDCK cells. In addition, recombinant IFN-λ1 inhibited the proliferation of MDCK cells slightly. These effects were observed in a dose-dependent manner. These results indicate that canine IFN-λ1 has antiviral effect, and suggest the potential applicability of canine IFN-λ1 as a therapeutic agent.
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http://dx.doi.org/10.1016/j.vetimm.2013.09.013DOI Listing
November 2013

Tumor suppressor candidate TUSC3 expression during rat testis maturation.

Biosci Biotechnol Biochem 2013 7;77(10):2019-24. Epub 2013 Oct 7.

Laboratory of Veterinary Biochemistry, Faculty of Agriculture, Tottori University.

Analysis of microarray data obtained by comparing gene expression between 2-week-old infant and 7-week-old mature SD rat testes revealed novel targets involved in tumor suppression. Reverse-transcription polymerase chain reaction and Northern blotting indicated that Tusc3 gene expression was upregulated in the normal maturing testis and prostate and other organs such as the cerebrum and ovary. Tumor suppressor candidate 3 protein expression was detected in these same organs at a size of about 40 kDa, in accord with the predicted molecular size. In situ hybridization and immunohistochemistry showed that mRNA and protein localization were prevalent in the testis spermatocytes and interstitial cells such as the Leydig cells, as well as prostate epithelial cells. These data suggest that TUSC3 is deeply involved in spermatogenesis in the testis, inducing sperm differentiation and maturation, and plays a role in normal prostate development and tumor suppression.
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http://dx.doi.org/10.1271/bbb.130327DOI Listing
July 2014

Development of an ELISA using a recombinant P46-like lipoprotein for diagnosis of Mycoplasma pulmonis infection in rodents.

J Vet Med Sci 2014 Mar 20;76(2):151-7. Epub 2013 Sep 20.

Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan.

Mycoplasma pulmonis is one of the most prevalent bacterial pathogens that infects laboratory mice and rats. To develop an M. pulmonis-specific antigen for serological diagnosis, we cloned the cDNA of P46-like lipoprotein (P46L), an M. pulmonis putative periplasmic protein. P46L is a homolog of P46, an M. hyopneumoniae antigen. We produced recombinant P46L fused to glutathione S-transferase (GST) in Escherichia coli. Immunoblot analysis revealed that sera from Mycoplasma-infected mice and rats contained anti-P46L antibodies. We developed an ELISA using the recombinant P46L-GST protein as an antigen. Thirteen of the 14 samples from rats naturally infected with M. pulmonis were determined to be positive according to the commercial ELISA (MONILISA Myco) and positive by our ELISA. Furthermore, 18/19 samples from mice experimentally infected with M. pulmonis were positive using our P46L-GST ELISA. In contrast, only 8/19 samples from infected mice were positive by the commercial ELISA. Our results indicate that P46L-GST was an appropriate antigen for developing a serological test to determine M. pulmonis infection in laboratory mice and rats.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982827PMC
http://dx.doi.org/10.1292/jvms.13-0308DOI Listing
March 2014
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