ARNTL Transcription Factors ; deficiency ; metabolism ; Animals ; Cyclic AMP ; metabolism ; Gluconeogenesis ; Glucose ; biosynthesis ; HEK293 Cells ; Histone Deacetylases ; metabolism ; Humans ; Liver ; metabolism ; Mice ; Mice, Knockout

Cyclic amp receptor protein (CRP) is a global transcriptional factor in many prokaryotes, capable of governing nearly half of the total genes in Escherichia coli. Through the method of error-prone PCR or DNA shuffling, we can first obtain CRP mutant library and then get the expected cell phenotype with enhanced resistance. In this article, we reviewed the following desired phenotype: enhanced tolerance towards oxidative stress, improved osmotolerance, enhanced organic solvent (toluene) tolerance, improved acetate tolerance of E. coli fermentation and improved ethanol tolerance during bio-ethanol production. We then concluded that CRP can also be applied in other host cells to get desired phenotypes. Last, we predicted potential applications of mutant CRP transcriptional factor.
Cyclic AMP Receptor Protein ; biosynthesis ; DNA Shuffling ; Escherichia coli ; metabolism ; Fermentation ; Metabolic Engineering

In this study, the essential oil from lotus flower extract, including petals and stamens, was assessed with regard to its effects on melanogenesis in human melanocytes. The lotus flower essential oil was shown to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner. The lotus flower essential oil induced the expression of tyrosinase, microphthalmia-associated transcription factor M (MITF-M), and tyrosinase-related proten-2 (TRP-2) proteins, but not tyrosinase mRNA. Moreover, it increased the phosphorylation of ERK and cAMP response element binding protein (CREB). In order to verify the effective components of the lotus flower oil, its lipid composition was assessed. It was found to be comprised of palmitic acid methyl ester (22.66%), linoleic acid methyl ester (11.16%), palmitoleic acid methyl ester (7.55%) and linolenic acid methyl ester (5.16%). Among these components, palmitic acid methyl ester clearly induced melanogenesis as the result of increased tyrosinase expression, thereby indicating that it may play a role in the regulation of melanin content. Thus, our results indicate that lotus flower oil may prove useful in the development of gray hair prevention agents or tanning reagents.
Blotting, Western ; Cell Proliferation ; Cyclic AMP/metabolism ; Cyclic AMP Response Element-Binding Protein/genetics/metabolism ; Flowers/*chemistry ; Gas Chromatography-Mass Spectrometry ; Humans ; Intramolecular Oxidoreductases/genetics/metabolism ; Lotus/*chemistry ; Melanins/*biosynthesis ; Melanocytes/*drug effects/metabolism ; Microphthalmia-Associated Transcription Factor/genetics/metabolism ; Monophenol Monooxygenase/genetics/metabolism ; Phosphorylation ; Plant Oils/*pharmacology ; RNA, Messenger/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/cytology/drug effects/metabolism

OBJECTIVETo explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression.

METHODSWashed platelets from healthy volunteers were incubated with PKA inhibitor. The N-terminal fragment of GP I balpha (glycocalicin, GC) in the supernatant of platelet suspensions was detected by Western blot and GP I balpha surface expression by flow cytometry. Calpain activity was determined by cytoskeletal proteins proteolysis and calpain surface expression by flow cytometry. The effect of PKA inhibitor on ristocetin-induced platelet aggregation was measured by platelet aggregometer.

RESULTSAfter PKA was inhibited in washed platelets, GP I balpha was cleaved and released to the supernatant, which significantly decreased the surface expression of GP I balpha (P < 0.05). The event was suppressed by pre-treatment with various calpain inhibitors, indicating that PKA inhibitor-mediated shedding was calpain dependent. The actin-binding protein (ABP) and talin proteolysis demonstrated that calpain was activated by PKA inhibitor and expressed on the platelet membrane. Ristocetin-induced aggregation was inhibited by PKA inhibitor.

CONCLUSIONPKA inhibition results in calpain-dependent GP I balpha shedding, which thus reduces GP I balpha surface expression and GP I balpha-dependent platelet aggregation. These results might provide a view to develop new drugs for thrombotic diseases.

Blood Platelets ; drug effects ; Calpain ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; antagonists & inhibitors ; Flow Cytometry ; Humans ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology ; Platelet Glycoprotein GPIb-IX Complex ; biosynthesis

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 micrometer) and bell-shaped. The concentrations of ATP, ATP-gammaS, ADP, and ADP-beta S that showed maximal IL-10 release were 100, 10, 100, and 100 micrometer respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP) = dATP > 2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), alpha,beta-methylene ATP (alpha,beta-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca2+ release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down- regulated by either MRS2179 (a P2Y1 antagonist) or 5'-AMPS (a P2Y11 antagonist), indicating that both the P2Y1 and P2Y11 receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-gammaS (100 micrometer) was restored by TNP-ATP (an antagonist of the P2X1, P2X3, and P2X4 receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y12 receptor antagonist) or pertusis toxin (PTX; a Gi protein inhibitor), indicating that the P2X1, P2X3, P2X4 receptor group, or the P2Y12 receptor, negatively modulate the P2Y11 receptor or the P2Y1 receptor, respectively.
Adenosine Diphosphate/analogs & derivatives/pharmacology ; Adenosine Triphosphate/analogs & derivatives/*pharmacology ; Adenylate Cyclase/antagonists & inhibitors ; Animals ; Calcium/metabolism ; Chelating Agents/pharmacology ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors ; Enzyme Inhibitors/pharmacology ; Extracellular Space/drug effects/*metabolism ; Gene Expression Regulation/drug effects ; Interleukin-10/*biosynthesis ; Microglia/*drug effects/enzymology/*metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor Cross-Talk/*drug effects ; Receptors, Purinergic P2/agonists/antagonists & inhibitors/genetics/*metabolism ; Thionucleotides/pharmacology

Chemokines and chemokine receptors play a role in migration of circulating leukocytes to the region of inflammation. Human LZIP is an uncharacterized transcription factor and is known to participate in leukotactin (Lkn)-1/CCL15-induced cell migration. We investigated the role of human LZIP in expression of CC chemokine receptors (CCRs) and its involvement in monocyte migration. RNase protection analysis showed that LZIP increased mRNA expression of CCR2 and CCR1 in THP-1 cells. Surface expressions of both CCR2 and CCR1 were also increased by LZIP. Results from an electrophoretic mobility shift assay showed that LZIP binds to the C/EBP element in the CCR2 promoter. LZIP also enhanced the chemotactic activities of monocyte chemoattractant protein-1/CCL2 and Lkn-1. These results suggest that LZIP regulates expression of chemokine receptors that are involved in monocyte migration.
Atherosclerosis/drug therapy/etiology ; CCAAT-Enhancer-Binding Proteins/genetics/immunology/*metabolism ; Cell Line, Tumor ; Cell Movement/drug effects/*physiology ; Chemokine CCL2/*pharmacology ; Chemokines, CC/pharmacology ; Cyclic AMP Response Element-Binding ; Humans ; Macrophage Inflammatory Proteins/pharmacology ; Monocytes/drug effects/metabolism ; Promoter Regions, Genetic ; Protein Binding ; RNA, Messenger/analysis ; Receptors, CCR1/biosynthesis/genetics ; Receptors, CCR2/*biosynthesis/genetics ; Transcriptional Activation/drug effects ; Transfection ; Transgenes

To investigate the function of testis sperm binding protein (TSBP) in sperm capacitation and acrosome reaction, the effect of the recombinant TSBP on the activity of protein kinase A was detected in the transfected cell line. With the use of prokaryotic expressing plasmid pGEX-5X-1/tsbp as template, the novel gene tsbp was amplified by PCR and a eukaryotic expressing vector pcDNA3.1/myc-His(-)B/tsbp was constructed. DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pcDNA3.1/myc-His(-)B/tsbp had been constructed successfully. After the recombinant plamid being transfected into HEK293 cells, Western blot verified the expression of tsbp. Fusion protein His6-TSBP was purified from the cell lysate by immobilized metal-ion affinity chromatography (IMAC). Radioautograph revealed a higher PKA activity in the transfected HEK293 cells than in the control group, which indicates that TSBP can increase the activity of PKA.
Acrosome Reaction ; drug effects ; Cell Line ; Cyclic AMP-Dependent Protein Kinases ; drug effects ; Humans ; Male ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Seminal Plasma Proteins ; biosynthesis ; genetics ; pharmacology ; Sperm Capacitation ; drug effects

Serotonin receptor subtype 6 (5-HT(6)) is a neurotransmitter receptor, which is involved in various brain functions such as memory and mood. It mediates signaling via the interaction with a stimulatory G-protein. Especially, the third intracellular loop (iL3) of 5-HT(6) and the alpha subunit of stimulatory G protein (Galpha(s)) are responsible for the signaling process of 5-HT(6). Chemical compounds that could inhibit the interaction between the iL3 region of 5-HT(6) and Galpha(s) were screened from a chemical library consisted of 5,600 synthetic compounds. One of the identified compounds bound to Galpha(s) and effectively blocked the interaction between Galpha(s) and the iL3 region of 5-HT(6). The identified compound was further shown to reduce the serotonin-induced accumulation of cAMP in 293T cells transformed with 5-HT(6) cDNA. It also lowered the Ca2+ efflux induced by serotonin in cells expressing 5-HT(6) and chimeric Galpha(s5/q). These results indicate that the interaction between the iL3 of 5-HT(6) and Galpha(s) can be exploited for screening of regulatory compounds against the signaling pathway of 5-HT(6).
Animals ; Calcium/metabolism ; Cell Line ; Cephalosporins/*pharmacology ; Cricetinae ; Cricetulus ; Cyclic AMP/biosynthesis ; GTP-Binding Protein alpha Subunits, Gs/antagonists & inhibitors/*metabolism ; Humans ; Receptors, Serotonin/*drug effects/metabolism/*physiology ; Serotonin/pharmacology ; Serotonin Antagonists/pharmacology ; Signal Transduction

Pigmentation may result from melanocyte proliferation, melanogenesis, migration or increases in dendricity. Recently, it has been reported that secreted phospholipase A2(sPLA2) known as a component of bee venom (BV), stimulates melanocyte dendricity and pigmentation. BV has been used clinically to control rheumatoid arthritis and to ameliorate pain via its anti-inflammatory and antinociceptive properties. Moreover, after treatment with BV, pigmentation around the injection sites was occasionally observed and the pigmentation lasted a few months. However, no study has been done about the effect of BV on melanocytes. Thus, in the present study, we examined the effect of BV on the proliferation, melanogenesis, dendricity and migration in normal human melanocytes and its signal transduction. BV increased the number of melanocytes dose and time dependently through PKA, ERK, and PI3K/Akt activation. The level of cAMP was also increased by BV treatment. Moreover, BV induced melanogenesis through increased tyrosinase expression. Furthermore, BV induced melanocyte dendricity and migration through PLA2activation. Overall, in this study, we demonstrated that BV may have an effect on the melanocyte proliferation, melanogenesis, dendricity and migration through complex signaling pathways in vitro, responsible for the pigmentation. Thus, our study suggests a possibility that BV may be developed as a therapeutic drug for inducing repigmentation in vitiligo skin.
Animals ; Base Sequence ; Bee Venoms/*pharmacology ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Cyclic AMP/metabolism ; DNA Primers/genetics ; Forskolin/pharmacology ; Gene Expression/drug effects ; Humans ; Melanins/biosynthesis ; Melanocytes/cytology/*drug effects/physiology ; Microphthalmia-Associated Transcription Factor/biosynthesis/genetics ; Monophenol Monooxygenase/biosynthesis/genetics ; Signal Transduction/drug effects

OBJECTIVETo compare the differences of expressions of adenylyl cyclase (AC) and phosphodiesterase (PDE) in ejaculated spermatozoa between healthy volunteers and the patients with asthenospermia.

METHODSEjaculated spermatozoa were collected from healthy volunteers and the patients with asthenospermia. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of AC and PDE subtypes in human spermatozoa. The concentrations of cAMP and cGMP in the samples were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSCompared with healthy volunteers, expression of sAC mRNA and concentration of cAMP were significantly decreased in the patients with asthenospermia (P < 0.01) , while the expression of PDE4C mRNA was significantly increased at the same time (P <0.01). There were no marked differences in the expression of ACIII mRNA and concentration of cGMP between the two groups.

CONCLUSIONThe sAC down-regulation and PDE4C up-regulation are possible reasons for asthenospermia.

Adenylyl Cyclases ; biosynthesis ; Asthenozoospermia ; metabolism ; Cyclic AMP ; metabolism ; Humans ; Male ; Phosphoric Diester Hydrolases ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatozoa ; metabolism