• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1216-1220
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• 2008

Sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2,199 bp sesquiterpene alcohol (germacradienol) synthase-encoding gene from the germacradienol synthase/terpene cyclase gene cluster. The gene was named spterp13, and its putative function is as a germacradienol synthase/terpene cyclase. The amino acid sequence of Spterp13 shows 66% identity with SAV2163 (GeoA) from S. avermitilis MA4680 and 65% identity with SCO6073 from S. coelicolor A3(2), which produces germacradienol/geosmin. The full-length recombinant protein was heterologously expressed as a his-tagged fusion protein in Escherichia coli, purified, and shown to catalyze the $Mg^{2+}$-dependent conversion of farnesyl diphosphate to the germacradienol, which was verified by gas chromatography/mass spectrometry.

• Parasites, Hosts and Diseases
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• v.60 no.1
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• pp.7-14
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• 2022

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.15-23
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• 2006

Objective: Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel hypothalamic neuropeptide, has been suggested to play a role in ovarian folliculogenesis. The present study evaluated the effect of PACAP on the growth of preantral follicles. Methods: Preantral follicles were mechanically isolated from ovaries of 21-day-old rats and cultured in groups for 3 days in serum-free medium in the absence or presence of PACAP-38 ($10^{-6}M$). Results: Treatment with PACAP-38 resulted in an increase in follicle diameter by 75% whereas treatment with follicle stimulating hormone (FSH) increased follicle diameter by 65%. PACAP-38 treatment enhanced the granulosa cell proliferation as measured by thymidine incorporation analysis. Furthermore, the production of progesterone by cultured granulosa cells and GFSHR-17 cell line was stimulated by PACAP-38. Interestingly, PACAP enhanced FSH action on stimulation of SF-1 and aromatase gene expression. Conclusion: The present results demonstrate that PACAP stimulated preantral follicle growth by potentiating proliferation and by stimulating steroidogenesis.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.343-351
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• 2001

This study was undertaken to investigate the mechanism of lipopolysaccharide (LPS) and nitric oxide (NO) as a regulator of vascular smooth muscle cell (VSMC) proliferation. VSMC was primarily cultured from rat aorta and confirmed by the immunocytochemistry with anti-smooth muscle myosin antibody. The number of viable VSMCs were counted, and lactate dehydrogenase (LDH) activity was measured to assess the degree of cell death. Concentrations of nitrite in the culture medium were measured as an indicator of NO production. LPS was introduced into the medium to induce the inducible nitric oxide synthase (iNOS) in VSMC, and Western blot for iNOS protein and RT-PCR for iNOS mRNA were performed to confirm the presence of iNOS. Inhibitors of iNOS and soluble guanylate cyclase (sGC), sodium nitroprusside (SNP) and L-arginine were employed to observe the action of LPS on the iNOS-NO-cGMP signalling pathway. LPS and SNP decreased number of VSMCs and increased the nitrite concentration in the culture medium, but there was no significant change in LDH activity. A cell permeable cGMP derivative, 8-Bromo-cGMP, decreased the number of VSMCs with no significant change in LDH activity. L-arginine, an NO substrate, alone tended to reduce cell count without affecting nitrite concentration or LDH level. Aminoguanidine, an iNOS specific inhibitor, inhibited LPS-induced reduction of cell numbers and reduced the nitrite concentration in the culture medium. LY 83583, a guanylate cyclase inhibitor, suppressed the inhibitory actions of LPS and SNP on VSMC proliferation. LPS increased amounts of iNOS protein and iNOS mRNA in a concentration-dependent manner. These results suggest that LPS inhibits the VSMC proliferation via production of NO by inducing iNOS gene expression. The cGMP which is produced by subsequent activation of guanylate cyclase would be a major mediator in the inhibitory action of iNOS-NO signalling on VSMC proliferation.

• The Korean Journal of Zoology
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• v.29 no.3
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• pp.181-189
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• 1986

The present study was undertaken to investigate whether the known adenylate cyclase activators, forskolin and cholera toxin, would affect the germinal vesicle breakdown (GVBD) and the production of cAMP in mouse oocytes in vitro. To do this, in vitro oocyte culture method and adenylate cyclase assay were employed. In response to different concentrations of forskolin (20 to 80 $\\mu$g/ml) added to a culture medium, the percentage of GVBD significantly decreased (56 to 31%) in a dose-dependent manner as compared to that of control (63%). This inhibitory phenomenon by forskolin was reversible since the rate of GVBD was returned to the control level when the oocytes were transferred to a control medium following exposure to forskolin (80 $\\mu$g/ml). Treatment of cholera toxin (10 to 1, 000 ng/ml) was, however, ineffective in suppressing GVBD. When forskolin (10 to 80 $\\mu$g/ml) was added to the mouse oocyte extracts, cAMP production significantly increased by 5 to 18 fold, whereas cholera toxin (10 to 1, 000 ng/ml) was no longer effective. In addition, treatment of guanidyl-imidodiphosphate (GppNHp, 100 $\\mu$M), which is an activator of the regulatory unit of adenylate cycleas, with forskolin did not exhibit any changes in cAMP production as compared to that induced by forskolin alone. Neither cholera toxin nor cholera toxin plus GppNHp (100 $\\mu$M) exhibited any differences in mouse oocytes. From the above results, the suppression of GVBD by forskolin may be mediated by a high level of intracellular cAMP in mouse oocytes. It appears that the changes in intracellular cAMP level may an important role in the mouse oocyte maturation.

• Journal of Life Science
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• v.24 no.12
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• pp.1330-1338
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• 2014

In Mycobacterium tuberculosis H37Rv 16 adenylyl cyclase (AC) genes have been identified, while 10 AC genes have been found in non-pathogenic Mycobacterium smegmatis. Expression of 6 AC genes (MSMEG_0218, MSMEG_3243, MSMEG_3780, MSMEG_4279, MSMEG_4477, and MSMEG_6154) among 10 AC genes in M. smegmatis was increased when M. smegmatis was subjected to hypoxic growth conditions. On the other hand, only MSMEG_3780 and MSMEG_4279 were slightly induced in the presence of NO. The cAMP levels in cells and culture media were 450- and 9.8-fold increased, respectively, when M. smegmatis was grown under hypoxic conditions relative to those grown aerobically. Intracellular levels of cAMP were increased 5.8-fold on the exposure to NO. The DevSR two-component system is known to be involved in the induction of many genes whose expression is induced under hypoxic conditions and in the presence of NO. Expression of 6 hypoxically induced AC genes was observed to be induced in a devR deletion mutant grown under hypoxic conditions, indicating that the induction of the 6 AC genes under hypoxic conditions is independent of the DevSR two-component system. In order to identify a trans-acting regulatory element that is pertinent in the hypoxic induction of MSMEG_3780, ligand-fishing analysis was performed using the upstream DNA of MSMEG_3780 and MSMEG_5136 protein was identified.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.5
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• pp.435-440
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• 2015

This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive $K^+$ channel blocker). However, neither $N^G$-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-${\alpha}$]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive $K^+$ channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.

• The Korean Journal of Pharmacology
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• v.28 no.2
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• pp.129-136
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• 1992

As it has been reported that the depolarization-induced acetylcholine (ACh) release is modulated by activation of presynaptic $A_1-adenosine$ heteroreceptor in hippocampus and various lines of evidence indicate the involvement of adenylate cyclase system in $A_1-adenosine$ post-receptor mechanism in hippocampus, it was attempted to delineate the role of adenylate cyclase system in the $A_1-receptor-mediated$ control of ACh release in this study. Slices from rat hippocampus were incubated with $[^3H]-choline$ and the release of the labelled products was evoked by electrical stimulation $(3\;Hz,\;5\;Vcm^{-1},\;2\;ms,\;rectangular\;pulses)$, and the influence of various agents on the evoked tritium-outflow was investigated. $N^6-cyclopentyladenosine$ (CPA), a specific $A_1-adenosine$ receptor agonist, in concentrations ranging from 0.1 to $10\;{\mu}M$, decreased the $[^3H]-ACh$ release in a dose-dependent manner without the changes of basal rate of release. 8-cyclopentyl-1,3-dipropylxanthine $(DPCPX,\;1{\sim}10\;{\mu}M)$, a selective $A_1-receptor$ antagonist, increased the $[^3H]-ACh$ release in a dose-related fashion with slight increase of basal tritium-release. And the CPA effects were significantly inhibited by DPCPX $(2\;{\mu}M)$ pretreatment and the dose-response curve produced by CPA was shifted to the right. The responses to N-ethylmaleimide $(NEM,\;10\;&\;30\;{\mu}M)$, a SH-alkylating agent of G-protein, were characterized by increments of the evoked ACh-release and the basal release, and the CPA effect were completely abolished by NEM pretreatment. Forskolin, a specific adenylate cyclase activator, in concentrations ranging from 0.3 to $10\;{\mu}M$, increased the evoked ACh-release in a dose-dependent manner and the CPA effects were inhibited by forskolin. These results indicate that the $A_1-adenosine$ heteroreceptor plays an important role in ACh-release via nucleotide-binding protein Gi in the rat hippocampus and that the adenylate cyclase system might be participated in this process.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.415-422
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• 2005

library of the mutants was prepared by transposon mutagenesis of the Mycobacterium bovis BCG. We screened this library for the resistance to an anti-tuberculosis antibiotic, PA-824. Most of the mutants resistant to the PA-824 were not able to synthesize the coenzyme $F_{420}$ which is normally produced by the wild type M. bovis BCG strains. HPLC analysis of the cellular extract showed that one of those mutants which lost the ability to synthesize $F_{420}$ still produced F0. The insertion site of the transposon in this mutant was determined by an inverse PCR and the transposon was found to be inserted in the Rv2435c open reading frame (ORF). Rv2435c ORF is predicted to encode an 80.3 kDa protein. Rv2435c protein appears to be bound to the cytoplasmic membrane, its N-terminal present in the periplasm and C-terminal in the cytoplasm. The C-terminal portion of this protein is highly homologous with the adenylyl cyclases of both prokaryotes and eukaryotes. There are 15 ORFs which have homology with the class III AC proteins in the genome of the M. tuberculosis and M. bovis. Two of those, Rv1625c and Rv2435c, are highly homologous with the mammalian ACs. We cloned the cytoplasmic domain of the Rv2435c ORF and expressed it with six histidine residues attached on its C-terminal in Escherichia coli to find out if this protein is a genuine AC. Production of that protein in E. coli was proved by purifying the histidine-tagged protein by using the Ni-NTA resin. This protein, however, failed to complement the cya mutation in E. coli, indicating that this protein lacks the AC activity. All of the further attempts to convert this protein to a functional AC by a mutagenesis with UV or hydroxylamine, or construction of several different fusion proteins with Rv1625c failed. It is, therefore, possible that Rv2435c protein might affect the conversion of F0 to $F_{420}$ not by synthesizing cAMP but by some other way.

• Journal of the Korean Chemical Society
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• v.37 no.1
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• pp.178-180
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• 1993