• YAKHAK HOEJI
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• v.34 no.4
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• pp.224-237
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• 1990

A single uniform population of specific, saturable, high affinity binding site of $[^3H]QNB$ guinuclidinyl benzilate(QNB) was identified in the rat cerebral microsomes. The Kd value(37.2 pM) for $[^3H]QNB$ calculated from the kinetically derived rate constants was in agreement with the Kd value(48.9 pM) determined by analysis of saturation isotherms at various receptor concentrations. Dimenhydrinate(DMH), histamine $H_1-blocker$, increased Kd value for $[^3H]QNB$ QNB without affecting the binding site concentrations and this effect resulted from the ability of DMH to slow $[^3H]QNB-receptor$ association. Pirenzepine inhibition curve of $[^3H]QNB$ binding was shallow(nH = 0.52) indicating the presence of two receptor subtypes with high ($M_1-site$) and low($M_2-site$) affinity for pirenzepine. Analysis of these inhibition curves yielded that 68% of the total receptor populations were of the $M_1-subtype$ and the remaining 32% of the $M_2-subtype$. Ki values for the $M_1-$ and $M_2-subtypes$ were 2.42 nM and 629.3 nM, respectively. Ki values for $H_1-blockers$ that inhibited $[^3H]QNB$ binding varied with a wide range ($0.02-2.5\;{\mu}M$). The Pseudo-Hill coefficients for inhibition of $[^3H]QNB$ binding by most of $H_1-blockers$ examined except for oxomemazine inhibition of $[^3H]QNB$ binding were close to one. The inhibition curve for oxomemazine in competition with $[^3H]QNB$ was shallow(nH = 0.74) indicating the presence of two receptor populations with different affinities for this drug. The proportion of high and low affinity was 33:67. The Ki values for oxomemazine were $0.045{\pm}0.016\;{\mu}M$ for high affinity and $1.145{\pm}0.232\;{\mu}M$ for low affinity sites. These data indicate that muscarinic receptor blocking potency of $H_1-blockers$ varies widely between different drugs and that most of $H_1-blockers$ examined are nonselective antagonist for the muscarinic receptor subtypes, whereas oxomemazine might be capable of distinguishing between subclasses of muscarinic receptor.

• Journal of Life Science
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• v.23 no.3
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• pp.347-354
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• 2013

Innate immunity is the ability to differentiate infectious agents from self. The innate immune system is comprised of a complicated network of recognition and effector molecules that act together to protect the host in the early stage of an infectious challenge. Mannose-binding lectin (MBL or mannose-binding protein, MBP) belongs to the family of $Ca^{2+}$-dependent lectins (C-type lectin with a collagen-like domain), which are considered an important component of innate immunity. While it is associated with increased risk and severity of infections and autoimmunity, the most frequent immuno-deficiency syndrome was reported to be low MBL level in blood. Deficiency of human MBL is caused by mutations in the coding region of the MBL gene. Rat homologue gene of human MBL gene was used to study functions of wild type and mutant MBL proteins. Although extensive studies have yielded the structural information of MBL, the functions of MBL, especially mutant MBL, still require investigation. We previously reported the cloning of rat wild-type MBL gene and the production of a truncated form of MBL protein and its antibody. Here, we present the cloning of mutant MBL cDNA in collagen-like domain (R40C, G42D, and G45E) using site-directed mutagenesis and differential behaviors of wild type and mutant MBL in cells. The major difference between wild type and mutant MBL was that while wild type MBL was secreted, mutant MBL was inhibited for secretion, retained in endoplasmic reticulum, and still functioned as a lectin.

• Animal Bioscience
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• v.34 no.10
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• pp.1579-1589
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• 2021

Objective: This study was conducted to generate single stranded DNA oligonucleotides with selective affinity to bovine spermatozoa, assess its binding potential and explore its potential utility in trapping spermatozoa from suspensions. Methods: A combinatorial library of 94 mer long oligonucleotide was used for systematic evolution of ligands by exponential enrichment (SELEX) with bovine spermatozoa. The amplicons from sixth and seventh rounds of SELEX were sequenced, and the reads were clustered employing cluster database at high identity with tolerance (CD-HIT) and FASTAptamer. The enriched nucleotides were predicted for secondary structures by Mfold, motifs by Multiple Em for Motif Elicitation and 5' labelled with biotin/6-FAM to determine the binding potential and binding pattern. Results: We generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78,098 and 21,196 reads in the top ten clusters and FASTAptamer identified 2,195 and 4,405 unique sequences in the top three clusters from the sixth and seventh rounds, respectively. The identified oligonucleotides formed secondary structures with delta G values between -1.17 to -26.18 kcal/mol indicating varied stability. Confocal imaging with the oligonucleotides from the seventh round revealed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail). Use of a 5'-biotin tagged oligonucleotide from the sixth round at 100 pmol with 4×10⁶ spermatozoa could trap almost 80% from the suspension. Conclusion: The binding patterns and ability of the identified oligonucleotides confirms successful optimization of the SELEX process and generation of aptamers to bovine spermatozoa. These oligonucleotides provide a quick approach for selective capture of spermatozoa from complex samples. Future SELEX rounds with X- or Y- enriched sperm suspension will be used to generate oligonucleotides that bind to spermatozoa of a specific sex type.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.263-273
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• 2002

This study was undertaken to evaluate the effects of $\beta$-mercaptoethanol ($\beta$-ME) on lipid peroxidation and fertilization ability in vitro by xanthine (X) - xanthine oxidase (XO) system in boar spermatozoa frozen-thawed. The boar spermatozoa were treated with X and/or XO, and the spermatozoa viability were measured by the eosin-nigrosin stain method. In control group, level of vitality in boar spermatozoa were higher than in medium with X, XO and X+XO groups. No significant differences, however, were observed under the all conditions. The percentage of spermatozoa that reached acrosome reaction were significantly (P<0.05) higher in sperm treated without that than with $\beta$-ME under the all conditions. On the other hand, when spermatozoa were inseminated in medium with X and/or XO, the penetration rates in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. The lipid peroxidation of sperm was evaluated on the basis of malondialdehyde (MDA) production. The MDA were higher in sperm treated without that than with $\beta$-ME under the above all conditions. However, significant differences were not observed between medium with and without $\beta$-ME. Sperm-SH group were higher detected in medium with that than without $\beta$-ME under the all conditions. The activity of sperm binding to Bona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were significantly (P<0.05) higher than in medium with X+XO groups. The sperm binding in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. These results suggest that addition of $\beta$-ME in X-XO system may play a positive role in improving of fertilization ability in vitro.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.327-337
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• 2001

This study investigated the effects of superoxide dismutase (SOD) on lipid peroxidation and fertilizing ability in vitro of boar spermatozoa frozen-thawed. The percentages of motile sperm were highest when SOD of 10 units/$m\ell$ was added to washing medium for spermatozoa. However, the rates of motile sperm were not significantly different in different concentrations of SOD. On the other hand, the motile rates of sperm washed with SOD were lower in sperm inculbated for 120 min than 30 min regardless of the different concentrations of SOD. The percentage of spermatozoa that reached acrosome reaction were increased with incubation periods prolonged. No significant differences, however, were observed in acrosome reaction rates between sperm incubated with and without SOD supplementation for 0, 60 and 120 min. When oocyies inseminated with different concentrations of SOD, the penetration rates were significantly (P＜0.05) higher in medium with 1 unit/$m\ell$ than 0, 10 and 100 units/$m\ell$ of SOD. However, the proportions of polyspermit oocytes were significantly (P＜0.05) lower in medium with 10 and 100 units/$m\ell$ than 0 unit/$m\ell$ of SOD. In another experiment, the sperm suspension were also treated with different concentrations of SOD and were assayed far sulfhydryl(-SH) group content. In the groups treated with 100 units/$m\ell$ of SOD, sperm-SH group were higher than another groups. However, sperm-SH group content were not siginificantly different in spermatozoa treated with different concentrations of SOD. Under the same conditions, the lipid peroxidation of sperm was evaluated on the basis of malondialdehyde production. The addition of SOD to sperm suspension decreased the formation or malondialdehyde. However, there were not significantly different in sperm treated with different concentrations of SOD. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. The sperm binding to zona pellucida were gradually increased with SOD concentrations added. The number of spermatozoa binded to zona pellucida were significantly (P＜0.05) higher in medium with 100 units/$m\ell$ than 0 units/$m\ell$ of SOD. These findings suggested that SOD cause an enhancement penetrarion ability and sperm zona binding in boar spermatozoa frozen-thawed.

• Korean Journal of Animal Reproduction
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• v.27 no.2
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• pp.153-161
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• 2003

This study has evaluated effect of the spermatozoa incubation on the glycosidase activity and fertilizing ability in vitro in the pig. To identify sperm glycosidases specific for sugar residues found in the zona pellucida of pig oocytes, the spermatozoa were treated experimentally and assayed for activities of $\alpha$-L-fucosidase, $\alpha$-D-mannosidase, $\beta$-D-galactosidase and N-acetyl-$\beta$-D-glucosaminidase ($\beta$-GlcNAc'ase). The glycosidases activity were higher in spermatozoa incubated for 2h than without incubation. The $\beta$-GlcNAc'ase activity was at least two-fold higher than other glycosidase regardless of spermatozoa incubation. In the same glycosidases, the activity had a tendency to increase as time of spermatozoa incubation was prolonged, but there were no differences in spermatozoa incubated during the various periods (4~24h). The percentages of spermatozoa that reached acrosome reaction were affected by glycosidases in the medium (P<0.05, for mannosidase), and were higher in spermatozoa with that than without incubation. On the other hand, the spermatozoa motility were decreased with incubation periods, but no effects by different glycosidases on the change of sperm motility during the various periods of incubation. In other experiment, the binding and penetration of pig spermatozoa were tested with oocytes matured in vitro in the presence of various glycosidase. The penetration rates were decreased with incubation of spermatozoa when oocytes were inseminated in medium with different glycosidases. These rates were higher in spermatozoa non-incubated than with incubation for 2h (P<0.05 for GlcNAc'ase; P<0.01 for control group). The sperm-zona binding rate in control group were higherthan in medium with glycosidases. In addition, the highest binding rate were obtained in medium with GlcNAc'ase. In all glycosidases, the sperm-zona binding rate in spermatozoa without incubation were higher than incubation for 2h. The significant differences were obtained in spermatozoa treated with $\alpha$-D-mannosidase (P<0.05). These results suggest that $\beta$-GlcNAc'ase is present mainly in the plasma membrane of pig spermatozoa. It was also shown that the glycosidase activity were increased in all glycosidases in spite of low sperm-zona binding rate and penetration rates by spermatozoa incubation.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.317-325
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• 2001

This study investigated the effect of ferrous sulfate (Fe$^{2+}$) and/or ascorbic acid (Asc) on fertilizing ability in vitro of frozen-thawed boar spermatozoa. Using chlortetracycline (CTC) fluorescence, the spermatozoa was treated in preincubation medium with control, Fe$^{2+}$(1 mM), Asc (0.5 mM) and Fe$^{2+}$Asc to assessed for acrosome reaction, and the oocyte penetration test to determine whether the Fe$^{2+}$ and/or Asc can promote the penetration ability in vitro. When frozen-thawed spermatozoa was washed with preincubation medium, there were significantly (P ＜ 0.05) more acrosome-reacted in medium with Fe$^{2+}$Asc (38%) than control (27%). The penetration rates were also significantly (P ＜ 0.05) higher in medium with Fe$^{2+}$Asc (76%) than control (55%). Next, the lipid peroxidation of sperm was evaluated on the basis of malondialdehyde production following same treatments. The addition of Fe$^{2+}$Asc to sperm suspension increases the formation of malondialdehyde. However, there were not significantly different under the all conditions. The sperm suspension were also treated with control, Fe$^{2+}$, Asc and Fe$^{2+}$/Asc and assayed for sulfhydry1(-SH) group content. In the Fe$^{2+}$/Asc group, sperm-SH group were higher than another groups. In spermatozoa treated with Fe$^{2+}$ and/or Asc, however, no changes in sperm -SH-groups were detected when compared to controls. In another experiment, the activity of sperm binding to zona pellucida was evaluated through binding to salt-stored porcine oocytes. In control and Asc treatment groups, sperm binding to zona pellucida were significantly (P ＜ 0.05) higher than in medium with Fe$^{2+}$. On the other hand, there is not a significant increase in binding to zona pellucida with spermatozoa treated by Fe$^{2+}$/Asc. In summary, the present study suggests that Fe$^{2+}$/Asc causes an enhancement in fertilizing ability that is associated with penetration rate increased without change of spermatozoa binding capacity to homologous zona pellucida.o homologous zona pellucida.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.23-29
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• 1996

According to the traditional receptor model, competitive antagonists share with agonists the ability to bind to a common site on receptors, but they are different from agonist in that they cannot trigger the biological response-i.e., they lack intrinsic efficacy. Recent findings extend the model by indicating that not all antagonists display an intrinsic efficacy of zero but that some display 'inverse agonism'. In the present study we studied the inverse agonism at $A_1$ adenosine receptors in membranes prepared from rat cerebral cortex. Eight commercially available $A_1$ adenosine receptor antagonists (CGS-15943, ADPX, CPT, DPCPX, DPX, N-0840, PACPX and 8-PT) were screened for inverse agonism by measuring the extent of $[^{35}S]guanosine-5'-({\gamma}-thio)$ triphosphate $([^{35}S]GTP_{\gamma}S)$ binding to G proteins. The agonist-induced stimulation of $[^{35}S]GTP_{\gamma}S$ bindings was completely blocked in the presence of $A_1$ adenosine receptor antagonists. Under optimal conditions, two types of antagonists could be distinguished. Seven antagonists including DPCPX decreased the basal $[^{35}S]GTP_{\gamma}S$ binding in the absence of agonist, displaying inverse agonist activity. One (CGS-15943) had no effect on the basal bindings. N-ethylmaleimide treatment reduced the basal bindings as well as agonist-mediated stimulation of $[^{35}S]GTP_{\gamma}S$ bindings, indicating that a substantial amount of this binding reflects an activated state of the C proteins. In good agreement with these findings, 0.1 mM GTP decreased the apparent affinity of the receptors for the agonist PIA, increased that for DPCPX, and had no effect on that for CGS-15943.

• Toxicological Research
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• v.8 no.2
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• pp.343-357
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• 1992

Tumor necrosis factor-${\alpha}$(TNF), a polypeptide hormone secreted primarily by activated macrophages, was originally identified on the basis of its ability to cause hemorrhagic necrosis and tumor regression in vivo. Subsequently, TNF has been shown to be an important component of the host responses to infection and cancer and may mediate the wasting syndrome known as cachexia. These systemic actions of TNF are reflected in its diverse effects on target cells in vitro. TNF initiates its diverse cellular actions by binding to specific cell surface receptors. Although TNF receptors have been identified on most of animal cells, regulation of these receptors and the mechanisms which transduce TNF receptor binding into cellular responses are not well understood. Therefore, in the present study, the mechanisms how TNF receptors are being regulated and how TNF receptor binding is being transduced into cellular responses were investigated in rat liver plasma membranes (PM) and ME-180 human cervical carcinoma cell lines. $^{125}I$-TNF bound to high ($K_d=1.51{\pm}0.35nM$)affinity receptors in rat liver PM. Solubilization of PM with 1% Triton X-100 increased both high affinity (from $0.33{\pm}0.04\;to\;1.67{\pm}0.05$ pmoles/mg protein) and low affinity (from $1.92{\pm}0.16\;to\;7.57{\pm}0.50$ pmoles/mg protein) TNF binding without affecting the affinities for TNF, suggesting the presence of a large latent pool of TNF receptors. Affinity labeling of receptors whether from PM or solubilized PM resulted in cross-linking of $^{125}I$-TNF into $M_r$ 130 kDa, 90 kDa and 66kDa complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, $^{125}I$-TNF binding to control and TNF-pretreated membranes were assayed. Specific binding was increased by pretreatment with TNF (P<0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF. As a next step, the post-receptor events induced by TNF were examined. Although the signal transduction pathways for TNF have not been delineated clearly, the actions of many other hormones are mediated by the reversible phosphorylation of specific enzymes or target proteins. The present study demonstrated that TNF induces phosphorylation of 28 kDa protein (p28). Two dimensional soidum dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) resolved the 28kDa phosphoprotein into two isoforms having pIs of 6.2 and 6.1. The pIs and relative molecular weight of p28 were consistent with those of a previously characterized mRNA cap binding protein. mRNA cap binding proteins are a class of translation initiation factors that recognize the 7-methylguanosine cap structure found on the 5' end of eukaryotic mRNAs. In vitro, these proteins are defined by their specific elution from affinity columns composed of 7-methylguanosine 5'-triphosphate($m^7$GTP)-Sepharose. Affinity purification of mRNA cap binding proteins from control and TNF treated ME-180 cells proved that TNF rapidly stimulates phosphorylation of an mRNA cap binding protein. Phosphorylation occurred in several cell types that are important in vitro models of TNF action. The mRNA cap binding protein phosphorylated in response to TNF treatment was purifice, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor-4E(eIF-4E). These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the diverse cellular actions of TNF.

• Journal of Photoscience
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• v.9 no.2
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• pp.201-204
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• 2002

Epidermal cells produce a panel of antioxidants as well as cytokines after UVB irradiation, which counteract reactive oxygen species, however, how these antioxidants might regulate melanogenesis is unclear. An important constituent of the cellular antioxidant buffering system which controls the redox state of proteins is thioredoxin (TRX), a 13-kD protein that catalyzes thiol-disulfide exchange reactions, regulates activation of transcription factors, and possesses several other biological functions similar to cytokines. TRX suppressed the UVB-induced production and secretion of $\alpha$-melanocyte stimulating hormone ($\alpha$-MSH) and of adrenocorticotropic hormone (ACTH), and also suppressed proopiomelanocortin (POMC) mRNA expression by normal human keratinocyte (KC)s. Further, L-cysteine, N-acetyl-cysteine, $\alpha$-tocopheryl ferulate showed suppressive effect on UVB-induced POMC mRNA expression. However, TRX released from UVB-irradiated KCs stimulated melanogenesis by up-regulating MSH receptor expression and its binding activity in melanocyte (MC)s. UVB-induced KC derived cytokines such as IL1, IL6, and ET1 upregulated MSH-receptor binding ability as well as MCl-R mRNA expression in cultured normal human MCs. MCl-R has a tendency to be upregulated by UVB-induced KC-derived cytokines as well as by direct UVB irradiation. These results suggest that antioxidants such as TRX suppresses UVB induction of POMC, but in the case of MCl-R, this gene can be mainly in the trend of upregulation by UVB-induced KC-derived factors including TRX.