Vitamin D deficiency is a common health issue around the world. We therefore evaluated the associations of semen quality with both serum and seminal plasma vitamin D levels and studied the mechanisms underlying these by incubating spermatozoa with 1,25(OH)₂D In vitro. Two hundred and twenty-two men were included in our study. Vitamin D was detected using an electrochemiluminescence method. Spermatozoa used for In vitro experiments were isolated by density gradient centrifugation. Positive relationships of serum 25(OH)D with semen volume and seminal plasma fructose were identified. Seminal plasma 25(OH)D level showed no relationship with serum 25(OH)D level, while it was inversely associated with sperm concentration and positively correlated with semen volume and sperm kinetic values. In vitro, sperm kinetic parameters increased after incubation with 1,25(OH)₂D, especially upon incubation for 30 min with it at a concentration of 0.1 nmol l^-1. Under these incubation conditions, the upward migration of spermatozoa increased remarkably with increasing adenosine triphosphate (ATP) concentration. The concentration of cyclic adenosine monophosphate (cAMP) and the activity of protein kinase A (PKA) were both elevated, and the PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89) reversed the increase of ATP production. The concentrations of cytoplasmic calcium ions and nicotinamide adenine dinucleotide (NADH) were both enhanced, while mitochondrial calcium uniporter (MCU) inhibitor, Ruthenium 360 (Ru360) did not reverse the increase of ATP production. Therefore, seminal plasma vitamin D may be involved in regulating sperm motility, and 1,25(OH)₂D may enhance sperm motility by promoting the synthesis of ATP both through the cAMP/PKA pathway and the increase in intracellular calcium ions.
Adenosine Triphosphate/metabolism* ; Adult ; Calcium/metabolism* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Humans ; Male ; Semen/metabolism* ; Semen Analysis ; Signal Transduction/physiology* ; Sperm Motility/physiology* ; Spermatozoa/metabolism* ; Vitamin D/pharmacology* ; Vitamin D Deficiency/blood* ; Wit and Humor as Topic ; Young Adult

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.
Animals ; Bucladesine/pharmacology ; Cell Line ; Colforsin/pharmacology ; Cyclic AMP/*metabolism ; DNA-Binding Proteins/chemistry/*genetics/metabolism ; *Gene Expression Regulation ; Humans ; Luciferases/analysis ; Neurons/*metabolism ; PC12 Cells ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins/analysis ; *Response Elements ; Transcription Factors/chemistry/*genetics/metabolism

Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J⋅cm(-2). Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J⋅cm(-2) significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J⋅cm(-2) showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.
Adenine ; analogs & derivatives ; pharmacology ; Adenylyl Cyclase Inhibitors ; Alkaline Phosphatase ; analysis ; genetics ; radiation effects ; Anthraquinones ; Bone Morphogenetic Protein 2 ; genetics ; Calcium ; metabolism ; radiation effects ; Cell Culture Techniques ; Cell Differentiation ; radiation effects ; Cell Line ; Cell Proliferation ; radiation effects ; Coloring Agents ; Core Binding Factor Alpha 1 Subunit ; genetics ; Cyclic AMP ; antagonists & inhibitors ; radiation effects ; Gene Expression ; radiation effects ; Humans ; L-Lactate Dehydrogenase ; analysis ; Lasers, Semiconductor ; Low-Level Light Therapy ; instrumentation ; Osteocalcin ; genetics ; Osteogenesis ; genetics ; radiation effects ; Periodontal Ligament ; cytology ; radiation effects ; Radiation Dosage ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Tetrazolium Salts ; Thiazoles

OBJECTIVETo study the expression and role of cyclic-AMP response binding protein (CREB) and Bcl-2 in children with acute leukemia.

METHODSNinety-two children with acute leukemia (leukemia group) and 30 children with non-hematologic malignancies (control group) were enrolled. The mRNA and protein expression of CREB and Bcl-2 in bone marrow mononuclear cells were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot.

RESULTSThe mRNA and protein expression of CREB and Bcl-2 in the leukemia group was significantly higher than that in the control group (p<0.01). There were no significant differences in the expression of CREB and Bcl-2 between acute lymphoblastic leukemia and acute myeloid leukemia subgroups. At the initial diagnosis, the mRNA and protein expression of CREB and Bcl-2 in children with extramedullary infiltration was higher than that in children without (p<0.05). In the leukemia group, the mRNA and protein expression of CREB and Bcl-2 in the complete remission subgroup was significantly lower than that in the non-complete remission subgroup (p<0.01). High mRNA expression of CREB and Bcl-2 in the leukemia group was positively correlated with peripheral blood leucocyte counts (r=0.62, 0.71 respectively, p<0.05). There was a positive correlation between mRNA and protein expression of CREB and Bcl-2 (r=0.75, 0.68 respectively; p<0.05).

CONCLUSIONSThe expression of CREB and Bcl-2 may be correlated with the pathogenesis and clinical prognosis of childhood leukemia, however, their expression may not be associated with the classification of acute leukemia.

Acute Disease ; Adolescent ; Bone Marrow Cells ; metabolism ; Child ; Child, Preschool ; Cyclic AMP Response Element-Binding Protein ; analysis ; genetics ; Female ; Humans ; Infant ; Leukemia ; metabolism ; Male ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; genetics ; RNA, Messenger ; analysis

OBJECTIVEThis study examined the effects of prenatal application of taurine on mRNA expression of protein kinase A cAMP response element binding protein (PKA-CREB) signal pathway and glial cell line derived neurotrophic factor (GDNF) in fetal rat brains of intrauterine growth restriction (IUGR).

METHODSPregnant rats were randomly divided into 4 groups: normal control, IUGR model, low dose (100 mg/kg x d) and high dose (300 mg/kg x d) taurine treatment IUGR (n = 5 each). IUGR was induced by food restriction throughout pregnancy. PKA, CREB and GDNF mRNA expression in brains of newborn rats was detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSPKA, CREB and GDNF mRNA expression in the IUGR model group was significantly higher than that in the normal control group (p<0.05). Compared with the IUGR model group, mRNA expression of PKA and CREB in both the low dose and high dose taurine treatment groups increased significantly (p<0.05); GDNF mRNA expression in the high dose taurine treatment group also increased significantly (p<0.01).

CONCLUSIONSTaurine can increase mRNA expression of PKA, CREB and GDNF in fetal rat brains of IUGR. This suggests that prenatal application of taurine may increase neurogenesis of the central nervous system and endogenous secretion of neurotrophic factors, thus providing neuroprotective effects.

Animals ; Brain ; drug effects ; Cyclic AMP Response Element-Binding Protein ; genetics ; physiology ; Cyclic AMP-Dependent Protein Kinases ; genetics ; physiology ; Female ; Fetal Growth Retardation ; metabolism ; Fetus ; drug effects ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Male ; Pregnancy ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Taurine ; pharmacology

OBJECTIVETo observe therapeutic effects and mechanisms at different acupoint groups for treatment of the depression rat.

METHODSFifty Wistar rats were randomly divided into a normal group, a model group, a body acupoint group, a scalp acupoint group and a Fluoxetine hydrochloride group, 10 cases in each group. Depression rat model was established by using chronic unpredictable mild stress stimulation combined with lonely breeding for 21 days. EA (2 Hz, 1 mA, 20 min) was given at "Taichong" (LR 3), "Neiguan" (PC 6) and "Zusanli" (ST 36) in the body point group and "Baihui" (GV 20), "Yintang" (EX-HN 3), "Sishencong" (EX-HN 1) in the head point group, once daily, and Fluoxetine hydrochloride was given to the Fluoxetine hydrochloride group by intragastric administration, 2 mg/kg, once daily, for 21 days. Body weight of the rat was recorded, and the sucrose-intake test and forced swimming test were conducted one day before the experiment and on the 7th, 14th and 21st day of the experiment, and the open field test was conducted one day before the experiment and on the 21st day. p-CREB expression in the hippocampus of the rats were observed on the 22nd day.

RESULTSAs compared with the model group, in the head point group and the Fluoxetine group, the crossing and rearing movement times, the relative volume of sucrose-intake and p-CREB expression in the hippocampus increased significantly, and the duration of immovability in forced swimming test shortened significantly (P<0.05). The relative volume of sucrose-intake increased and the duration of immovability in forced swimming test in the body point group significantly shortened (P<0.05), but the crossing and rearing movement times and p-CREB expression in the hippocampus did not insignificantly increase (P>0.05), with no significant differences among the treatment groups (P>0.05).

CONCLUSIONEA at the head acupoints can improve behavioral activities and increase positive neuron number of p-CREB in the hippocampus of the depression rat, and selection of acupoints is of important role in treatment of depression.

Acupuncture Points ; Animals ; Body Weight ; Cyclic AMP Response Element-Binding Protein ; analysis ; Depression ; metabolism ; psychology ; therapy ; Electroacupuncture ; Hippocampus ; chemistry ; Male ; Motor Activity ; Phosphorylation ; Random Allocation ; Rats ; Rats, Wistar ; Swimming

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

BACKGROUNDThe 5-hydroxytryptamine7 receptor (5-HT(7) receptor, 5-HT(7)R) plays an important role in the regulation of smooth muscle relaxation and visceral sensation and might be involved in the pathogenesis of the gastrointestinal dyskinesia, abdominal pain and visceral paresthesia in irritable bowel syndrome (IBS). The aim of this study was to investigate the role of the 5-HT(7) receptor in the pathogenesis of IBS.

METHODSA rat model of irritable bowel syndrome with diarrhea (IBS-D) was established by colonic instillation of acetic acid and restraint stress. A rat model with irritable bowel syndrome with constipation (IBS-C) was established by stomach irrigated with 0 - 4 degrees C cool water daily for 14 days. The content and distribution of 5-HT in the brain and gut were examined by immunohistochemistry and the mRNA expression of the 5-HT(7) receptor was determined by fluorescent quantitative reverse transcription polymerase chain reaction. The accumulation of cyclic adenosine monophosphate (cAMP) in all the same tissues was measured by radioimmunity.

RESULTSThe models of IBS were reliable by identification. The immunohistochemistry results showed that there were significantly more 5-HT positive cells in the IBS-D group than in the control group in the hippocampus, hypothalamus, jejunum, ileum, proximate colon and distal colon (P < 0.05), as well as more than were found in the IBS-C group in jejunum and ileum (P < 0.05). There were more 5-HT positive cells in the IBS-C group than in the control hippocampus, hypothalamus, ileum, proximate colon, and distal colon (P < 0.05). Real time-PCR results showed that the expression level of the 5-HT(7) receptor in both the IBS-C and IBS-D groups were enhanced compared with the control group in the hippocampus and hypothalamus (P < 0.05). The expression level of 5-HT(7) receptors in the IBS-C group was notably greater when compared with the controls in the ileum and colon (P < 0.05). The cAMP accumulation in the hippocampus and hypothalamus in both the IBS-C and IBS-D groups was higher than that in the control group (P < 0.01 and P < 0.05). The cAMP accumulation in the IBS-C group was higher than that in the control group in the proximal and distal colon (P < 0.05).

CONCLUSIONSThe increased 5-HT content in the brain and intestine is related to the IBS pathogenesis. The up-regulated expression of the 5-HT(7) receptor in the brain and colon might play an important role in the pathogenesis of IBS-C.

Animals ; Brain ; metabolism ; Cyclic AMP ; metabolism ; Disease Models, Animal ; Immunohistochemistry ; Intestines ; metabolism ; Irritable Bowel Syndrome ; etiology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Serotonin ; analysis ; genetics ; physiology ; Serotonin ; analysis

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.
Animals ; Aorta, Thoracic/cytology ; Blotting, Western ; Cattle ; Cell Culture Techniques ; Cell Extracts ; Cells, Cultured ; Comparative Study ; Cyclic AMP-Dependent Protein Kinases/analysis/*metabolism ; Endothelium, Vascular/cytology/*enzymology/*metabolism ; Nitric Oxide Synthase Type III/analysis/*metabolism ; Phosphorylation ; Precipitin Tests ; Research Support, Non-U.S. Gov't ; Stress, Mechanical ; Time Factors

OBJECTIVETo observe anti-arrhythmic effect of acupuncture pretreatment in the rat of myocardial ischemia and reperfusion (MIR) and to explore the role of cAMP and Gsa protein in beta-adrenergic receptor signaling.

METHODSMIR was produced by ligation and reperfusion of the left anterior descending coronary artery in the rat. Arrhythmic score, content of cAMP and Gsalpha protein in ischemic myocardium were compared among the normal control (NC), ischemia and reperfusion (IR), electroacupuncture (EA) and EA plus propranolol (EAP) groups.

RESULTSThe arrhythmic score in the IR group at 10 min after reperfusion was higher than the NC group (P < 0.01); in the EA group the score was decreased (P < 0.01 vs the IR group); the score in the EAP group was similar to the IR group, much higher than the EA group (P < 0.01). The similar results for the contents of cAMP and Gsalpha protein were found in the ischemic myocardium. It is suggested that EA pretreatment significantly attenuates the arrhythmic incidence rate and the enhancement of the contents of myocardial cAMP and Gsalpha protein induced by MIR, and the attenuating effect is significantly inhibited by the intraperitoneal pretreatment of propranolol, a specific beta-adrenoceptor antagonist.

CONCLUSIONPretreatment of EA can produce anti-arrhythmic effect in the rat of MIR, which is mediated by the post-receptor signaling pathway of beta-adrenergic receptor.

Acupuncture Therapy ; Animals ; Arrhythmias, Cardiac ; prevention & control ; Calcium ; metabolism ; Cyclic AMP ; analysis ; GTP-Binding Protein alpha Subunits, Gs ; analysis ; Male ; Myocardial Ischemia ; metabolism ; therapy ; Rats ; Rats, Wistar ; Receptors, Adrenergic, beta ; physiology ; Signal Transduction ; physiology