M2 Phenotype Macrophages Colocalize with Schwann Cells in Human Dental Pulp.

Macrophages are immune cells with high plasticity that perform many functions related to tissue injury and repair. They are generally categorized as 2 functional phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory and prohealing). To investigate the role of macrophages in human dental pulp, we examined the localization and distributional alterations of macrophages in healthy dental pulp as well as during the reparative process of pulp capping with mineral trioxide aggregate (MTA) and in cariously inflamed pulp of adult human teeth. We also quantified the populations of M1/M2 macrophages in healthy dental pulp by flow cytometric analysis. CD68+CD86+ cells (M1 phenotype) and CD68+CD163+ cells (M2 phenotype) were 2.11% ± 0.50% and 44.99% ± 2.22%, respectively, of 2.96% ± 0.41% CD68+ cells (pan-macrophages) in whole healthy dental pulp. Interestingly, M2 phenotype macrophages were associated with Schwann cells in healthy pulp, during mineralized bridge formation, and in pulp with carious infections in vivo. Furthermore, the M2 macrophages associated with Schwann cells expressed brain-derived neurotrophic factor (BDNF) under all in vivo conditions. Moreover, we found that plasma cells expressed BDNF. Coculture of Schwann cells isolated from human dental pulp and human monocytic cell line THP-1 showed that Schwann cells induced M2 phenotypic polarization of THP-1 cell-derived macrophages. The THP-1 macrophages that maintained contact with Schwann cells were stimulated, leading to elongation of their cell shape and expression of M2 phenotype marker CD163 in cocultures. In summary, we revealed the spatiotemporal localization of macrophages and potent induction of the M2 phenotype by Schwann cells in human dental pulp. M2 macrophages protect neural elements, whereas M1 cells promote neuronal destruction. Therefore, suppressing the neurodestructive M1 phenotype and maintaining the neuroprotective M2 phenotype of macrophages by Schwann cells may be critical for development of effective treatment strategies to maintain the viability of highly innervated dental pulp.

Possible antithrombotic effects of Angelica keiskei (Ashitaba).

Angelica keiskei Koidzumi (Ashitaba) is a large perennial herb that is native to the Pacific coast of Japan, and it has recently become popular as herbal medicine, dietary supplement and health food in Asian countries. The structures of various constituents isolated from Ashitaba such as chalcones, flavanones and coumarins have been precisely characterized, and many of them have bioactivities. A recent study clarified that Angelica keiskei exerts actions that lead to the prevention of thrombosis. Here, we introduce the possibility that ingesting Ashitaba could help to prevent thrombotic diseases.

Detection of bone marrow-derived fibrocytes in human dental pulp repair.

AIM: To explore the expression profile of CD45+/pro-collagen I+ fibrocytes in intact dental pulps as well as during wound healing in adult dental pulp tissue. METHODOLOGY: A total of 16 healthy permanent teeth were obtained from young patients (18 to 25 years) undergoing orthodontic treatment. Routine pulp capping with mineral trioxide aggregate (MTA) was performed under local anaesthesia to induce a mineralized barrier at the exposed surface. Teeth were extracted from patients after 7, 14 and 35 days. Sections of the extracted teeth were prepared and stained for various markers using indirect immunofluorescence. Fibrocytes were counted, and the data were statistically evaluated using the Dunnett test. RESULTS: In uninflammed pulp tissue, a pro-collagen I-positive reaction was detected in odontoblasts, as well as in perivascular cells. Most of the CD45-positive cells were negative for pro-collagen I in normal pulp tissue, whereas CD45+/pro-collagen I+ fibrocytes were detected 7 days after injury. At day 14, fibrocytes were recognized under the fibrous matrix in contact with MTA and had infiltrated into regions of new capillary formation, where the fibrocytes were positively stained for vascular endothelial growth factor. By 35 days, fibrocytes were few, coincident with the formation of dentine bridges. The number of fibrocytes peaked 7 days post-injury and decreased at 14 days. CONCLUSIONS: The presence of fibrocytes in human pulp wound healing was observed. The spatiotemporal distribution of fibrocytes suggests that fibrocytes are involved in the early stages of pulp wound healing, specifically by contributing to new blood vessel formation.

IL4Rα and ADAM33 as genetic markers in asthma exacerbations and type-2 inflammatory endotype.

BACKGROUND: Genetic markers of susceptibility to asthma exacerbations in adults remain unclear. OBJECTIVE: To identify genetic markers of asthma exacerbations, particularly in patients with type-2 inflammatory endotype. METHODS: In this observational study of patients enrolled in the Kinki Hokuriku Airway disease Conference multicenter study, frequency of exacerbations requiring systemic corticosteroids during 2 years after enrolment and associated risk factors was determined. For genetic marker analysis, interleukin-4 receptor α (IL4RA) rs8832 and a disintegrin and metalloprotease 33 (ADAM33) S_2 (rs528557), T_1 (rs2280091), T_2 (rs2280090), and V_4 (rs2787094) variants were included. Elevated serum periostin levels at enrolment (≥95 ng/mL, defined as type-2 inflammatory endotype) were considered in the analysis. RESULTS: Among 217 patients who were successfully followed up for 2 years after enrolment, 60 patients showed at least one asthma exacerbation during the 2 years. Airflow limitation (%FEV1 <80%) and recent exacerbations but not genetic variants were identified as risk markers of exacerbations. A total of 27 patients showed type-2 inflammatory endotype (serum periostin ≥95 ng/mL at enrolment) and subsequent exacerbations; risk factors in these patients were airflow limitation (odds ratio, 6.51; 95% confidence interval (CI): 2.37-18.6; P=.0003), GG genotype of IL4RA rs8832 (odds ratio, 4.01; 95% CI: 1.47-11.0; P=.007), and A allele of ADAM33 T_2 (odds ratio, 2.81; 95% CI: 1.05-7.67; P=.04) by multivariate analysis. In addition, GG genotype of IL4RA rs8832 was associated with type-2 endotype, whereas A allele of ADAM33 T_2 was associated with mixed type of eosinophilic/type-2 and neutrophilic inflammations. CONCLUSIONS AND CLINICAL RELEVANCE: IL4RA and ADAM33 variants may be risk markers of asthma exacerbations in type-2 inflammatory endotype. Precise endotyping may facilitate the identification of genetic risk markers of asthma exacerbations.

GaAlAs laser-induced pulp mineralization involves dentin matrix protein 1 and osteopontin expression.

OBJECTIVES: GaAlAs lasers induce pulp mineralization by promoting reparative dentinogenesis. This study analyzed the expression of dentin matrix protein 1 (DMP1) and osteopontin in GaAlAs laser-irradiated rat molars, to examine the hypothesis that these proteins play a role in the laser-induced reparative dentinogenic process. MATERIALS AND METHODS: The mesial surfaces of the upper first molars of 8-week-old Wistar rats were irradiated with a pulsed GaAlAs laser. After 1-14 days, mRNA expression of DMP1 and osteopontin in the coronal pulp was analyzed using real-time PCR. DMP1, osteopontin, and heat shock protein 25 (HSP25) were immunolocalized at 1-21 days. RESULTS: The pulp exhibited a degenerative zone in its mesial portion on days 1-3, and progressive formation of reparative dentin lined with HSP25-immunoreactive odontoblast-like cells, from day 7 onwards. DMP1 and osteopontin mRNA expression were significantly upregulated on days 1-7 and 3-7, respectively. From day 7 onwards, DMP1 and osteopontin immunoreactivity colocalized along the boundary between the primary and reparative dentin. CONCLUSION: GaAlAs laser irradiation of rat molars induced upregulated DMP1 and osteopontin mRNA expression in the coronal pulp, followed by the formation of reparative dentin and the colocalization of DMP1 and osteopontin immunoreactivity at the site at which this tissue first appeared.

Anti-platelet effects of chalcones from Angelica keiskei Koidzumi (Ashitaba) in vivo.

Angelica keiskei Koidzumi (Ashitaba) is a traditional folk medicine that is also regarded in Japan as a health food with potential antithrombotic properties. The ability of the major chalcones, xanthoangelol (XA) and 4-hydroxyderricin (4-HD) extracted from Ashitaba roots to inhibit platelet aggregation activity in vitro was recently determined. However, the anti-platelet activities of Ashitaba chalcones in vivo have remained unclear. The present study examines the anti-platelet effects of Ashitaba exudate and its constituent chalcones using mouse tail-bleeding models that reflect platelet aggregation in vivo. Ashitaba exudate and the major chalcone subtype XA, suppressed the lipopolysaccharide (LPS)-induced shortening of mouse tail bleeding. However, trace amounts of other Ashitaba chalcone subtypes including xanthoangelols B (XB), D (XD), E (XE) and F (XF) did not affect tail bleeding. These results suggest that the major chalcone subtype in Ashitaba, XA, has anti-platelet-activities in vivo.

GLCCI1 variant accelerates pulmonary function decline in patients with asthma receiving inhaled corticosteroids.

BACKGROUND: In steroid-naive patients with asthma, several gene variants are associated with a short-term response to inhaled corticosteroid (ICS) treatment; this has mostly been observed in Caucasians. However, not many studies have been conducted for other ethnicities. Here, we aimed to determine the relationship between the annual decline in forced expiratory flow volume in one second (FEV1 ) and the variant of the glucocorticoid-induced transcript 1 gene (GLCCI1) in Japanese patients with asthma receiving long-term ICS treatment, taking into account the effect of high serum periostin levels, a known association factor of pulmonary function decline and a marker of refractory eosinophilic/Th2 inflammation. METHODS: In this study, 224 patients with asthma receiving ICS treatment for at least 4 years were enrolled. The effects of single-nucleotide polymorphisms (SNPs) in GLCCI1, stress-induced phosphoprotein 1 (STIP1), and T gene on the decline in FEV1 of 30 ml/year or greater were determined. RESULTS: Besides the known contributing factors, that is, the most intensive treatment step, ex-smoking, and high serum periostin levels (≥95 ng/ml), the GG genotype of GLCCI1 rs37973, and not other SNPs, was independently associated with a decline in FEV1 of 30 ml/year or greater. When patients were stratified according to their serum periostin levels, the GG genotype of rs37973 was significantly associated with blood eosinophilia (≥250/µl) in the high serum periostin group. CONCLUSIONS: A GLCCI1 variant is a risk factor of pulmonary function decline in Japanese patients with asthma receiving long-term ICS treatment. Thus, GLCCI1 may be associated with response to ICS across ethnicities.

Effects of highly ripened cheeses on HL-60 human leukemia cells: antiproliferative activity and induction of apoptotic DNA damage.

To establish cheese as a dairy product with health benefits, we examined the multifunctional role of cheeses. In this report, we clarify whether different types of commercial cheeses may possess antiproliferative activity using HL-60 human promyelocytic leukemia cell lines as a cancer model. Among 12 cheese extracts tested, 6 (Montagnard, Pont-l'Eveque, Brie, Camembert, Danablue, and Blue) revealed strong growth inhibition activity and induction of DNA fragmentation in HL-60 cells. Based on the quantification of nitrogen contents in different cheese samples, a positive correlation between the ripeness of various cheeses and their antiproliferative activity tested in HL-60 cells was displayed. Four varieties of Blue cheese ripened for 0, 1, 2, or 3 mo demonstrated that the Blue cheese ripened for a long term was capable of causing the strong suppression of the cell growth and the induction of apoptotic DNA damage as well as nucleic morphological change in HL-60 cells. Collectively, these results obtained suggest a potential role of highly ripened cheeses in the prevention of leukemic cell proliferation.

COX-2 inhibition attenuates cough reflex sensitivity to inhaled capsaicin in patients with asthma.

BACKGROUND AND OBJECTIVE: Cyclooxygenase (COX) is an enzyme that converts arachidonic acid to prostanoids. There are two isoforms of COX, namely COX-1 and COX-2. COX-2 is highly inducible by several stimuli and is associated with inflammation. Recent studies have shown that COX-2 is upregulated in the airway epithelium of patients with asthma but little is known about the role it plays in cough, a common symptom of bronchial asthma. This study was designed to investigate the role of COX-2 in cough reflex sensitivity in patients with asthma. PATIENTS AND METHODS: The effect of etodolac, a potent COX-2 inhibitor, on cough response to inhaled capsaicin was examined in 17 patients with stable asthma in a randomized, placebo-controlled crossover study. Capsaicin cough threshold, defined as the lowest concentration of capsaicin eliciting 5 or more coughs, was measured as an index of airway cough reflex sensitivity. RESULTS: The geometric mean (geometric SEM) cough threshold was significantly increased after a 2-week treatment program with oral etodolac (200 mg twice a day) compared with placebo (36.7 [1.2] vs 21.6 [1.2] gM, P<.02). CONCLUSIONS: These findings indicate that COX-2 may be a possible modulator augmenting airway cough reflex sensitivity in asthmatic airways.

Circadian clock molecules CLOCK and CRYs modulate fibrinolytic activity by regulating the PAI-1 gene expression.

Disruptions of circadian rhythms are associated with the development of many disorders. However, whether a disruption of the circadian clock can cause anomalies of the hemostatic balance remains unknown. The present study examines coagulation and fibrinolytic activities in circadian clock mutants, a homozygous Clock mutant and Cry1/Cry2 double knockout (Cry1/2-deficient) mice. The euglobulin clot lysis time (ELT) showed circadian variations that peaked at 21:00 (early night) in wild-type mice, suggesting that fibrinolytic activity is lowest at this time. The ELT was continuously reduced in Clock mutants, while the ELT was significantly increased and did not differ between day and night (9:00 and 21:00) in Cry1/2-deficient mice. The prothrombin time (PT) and activated partial prothrombin time (APTT) were constant in all genotypes. To identify which factors cause the loss of ELT rhythm, we measured fibrinolytic parameters in Clock mutant and Cry1/2-deficient mice. The robust circadian fluctuation of plasma plasminogen activator inhibitor 1 (PAI-1) that peaked at early night was damped to trough levels in Clock mutant mice. On the other hand, PAI-1 levels in Cry1/2-deficient mice remained equivalent to the peak levels of those in wild-type mice at both 9:00 and 21:00. Circadian changes in plasma PAI-1 levels seemed to be regulated at the level of gene expression, because the plasma PAI-1 levels in Clock mutant and Cry1/2-deficient mice were closely correlated with the level of PAI-1 mRNA transcript in these mice. Plasma plasminogen and hepatic mRNA levels were not rhythmic in wild-type mice, and continuously higher in Clock mutant than in wild-type or Cry1/2-deficient mice. In contrast, the activity and mRNA levels of tissue type plasminogen activator (t-PA), plasma levels and mRNA levels of plasminogen, and plasma levels of alpha2 plasmin inhibitor (alpha2PI) in all genotypes were constant throughout the day. Coagulation parameters such as factor VII, factor X, prothrombin and fibrinogen remained constant throughout the day, and were not affected by clock gene mutations. These results suggest that circadian clock molecules play an important role in hemostatic balance by regulating the fibrinolytic systems.

CLOCK is involved in obesity-induced disordered fibrinolysis in ob/ob mice by regulating PAI-1 gene expression.

BACKGROUND: An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. AIM: The present study investigates whether the circadian clock component CLOCK is involved in obesity-induced PAI-1 elevation. METHODS: We examined plasma PAI-1 and mRNA expression levels in tissues from leptin-deficient obese and diabetic ob/ob mice lacking functional CLOCK protein. RESULTS: Our results demonstrated that plasma PAI-1 levels were augmented in a circadian manner in accordance with the mRNA expression levels in ob/ob mice. Surprisingly, a Clock mutation normalized the plasma PAI-1 concentrations in accordance with the mRNA levels in the heart, lung and liver of ob/ob mice, but significantly increased PAI-1 mRNA levels in adipose tissue by inducing adipocyte hypertrophy in ob/ob mice. The Clock mutation also normalized tissue PAI-1 antigen levels in the liver but not in the adipose tissue of ob/ob mice. CONCLUSION: These observations suggest that CLOCK is involved in obesity-induced disordered fibrinolysis by regulating PAI-1 gene expression in a tissue-dependent manner. Furthermore, it appears that obesity-induced PAI-1 production in adipose tissue is not closely related to systemic PAI-1 increases in vivo.

Adrenal gland-dependent augmentation of plasminogen activator inhibitor-1 expression in streptozotocin-induced diabetic mice.

BACKGROUND: Diabetes is associated with an excess risk of cardiac events, and one risk factor for infarction is an elevated level of plasminogen activator inhibitor-1 (PAI-1). OBJECTIVES AND METHODS: To evaluate whether the glucocorticoid hormones are involved in the diabetes-induced PAI-1 production, we examined expression profiles of PAI-1 mRNA in adrenalectomized (ADX) mice with streptozotocin (STZ)-induced diabetes. RESULTS: The diabetes-induced augmentation of plasma PAI-1 levels and PAI-1 mRNA expression in the heart and lungs was completely normalized in diabetic ADX mice. The glucocorticoid receptor antagonist RU486 significantly, but only partly suppressed PAI-1 induction in STZ-induced diabetic mice, suggesting that factors other than glucocorticoids are also involved in PAI-1 induction provoked by diabetes. CONCLUSION: Our results suggested that the adrenal gland plays a critical role in the progression of thrombosis in diabetic patients by inducing expression of the PAI-1 gene.

Hypocoagulable state of human preovulatory ovarian follicular fluid: role of sulfated proteoglycan and tissue factor pathway inhibitor in the fluid.

Ovulation accompanied by tissue damage can cause an increase in the level of tissue factor (TF) in the follicular fluid, triggering the extrinsic coagulation pathway. However, follicular fluid must block fibrin formation and maintain fluidity until the release of the oocyte at ovulation. The combination of sulfated proteoglycan, antithrombin, and TF pathway inhibitor (TFPI) appears to play a critical role in the hypocoagulability of human follicular fluid. When compared with plasma, folicular fluid differs markedly in the levels of a number of important coagulation proteins. Principal among these are 15-fold, 13-fold, and 3.7-fold increases in free TFPI, thrombin-antithrombin complex, and TF, respectively. The excessively prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) of human ovarian follicular fluid appear to be primarily due to high concentrations of sulfated proteoglycans, which accelerate the inactivation of thrombin and the anti-Xa activity of TFPI. Thus, heparitinase treatment shortened the clotting times of follicular fluid and reduced the inhibition of thrombin by the proteoglycan fraction combined with a fraction containing antithrombin. The remaining prolongation of APTT and PT may be caused by high levels of free TFPI in follicular fluid, which were confirmed by Northern blotting analysis, demonstrating TFPI mRNA expression by granulosa cells.

Menin, a gene product responsible for multiple endocrine neoplasia type 1, interacts with the putative tumor metastasis suppressor nm23.

Although the gene responsible for multiple endocrine neoplasia type 1 (MEN1) has been identified, the function of its gene product, menin, is unknown. To examine the biological role of the MEN1 gene, we searched for associated proteins with a yeast two-hybrid system using the MEN1 cDNA fragment as bait. On screening a rat fetal brain embryonic day 17 library, in which a high level of MEN1 expression was detected, we identified a putative tumor metastasis suppressor nm23/nucleoside diphosphate (NDP) kinase as an associated protein. This finding was confirmed by in vitro interaction assays based on glutathione S-transferase pull down experiments. The association required almost the entire menin protein, and several missense MEN1 mutations reported in MEN1 patients caused a loss of the binding activity for nm23. This result suggests that this interaction may play important roles in the biological functions of the menin protein, including tumor suppressor activity.

The orphan nuclear receptor NOR-1 interacts with the homeobox containing protein Six3.

Neuron-derived orphan receptor 1 (NOR-1) is a member of the NGFI-B subfamily within the nuclear receptor superfamily. In order to identify cofactors that associate with NOR-1 in the fetal forebrain, we tested a yeast two-hybrid system with the NOR-1 cDNA fragment lacking a transactivating domain as a bait. By screening of the rat fetal brain embryonic day 17 library, a rat homologue of Six3 was identified as an associated protein. We demonstrated that NOR-1 interacted with Six3 in yeast and in vitro, and the association was required for the DNA binding and AF2 domains of NOR-1. Regarding the other members of the family (NGFI-B and RNR-1), association with Six3 was not observed in yeast. In addition, cotransfection experiments with Six3 and NOR-1 indicated that Six3 had a negative activity against the transactivation by NOR-1 through the NBRE response element in a dose-dependent manner. The overlap in expression of NOR-1 and Six3 was mainly detected in the rat fetal forebrain on embryonic day 18. Thereafter, the expression of both genes diminished rapidly. These results suggest that a dimer consisting of a homeobox containing protein Six3 and transcriptional factor NOR-1 might regulate gene expression during the late stage of the fetal forebrain development. This study provides, after the association of Ftz and Ftz-F1 in Drosophila, another example of a dimer formation of a homeobox protein and an orphan nuclear receptor.

Highly increased plasma concentrations of the nicked form of beta(2) glycoprotein I in patients with leukemia and with lupus anticoagulant: measurement with a monoclonal antibody specific for a nicked form of domain V.

beta(2)-Glycoprotein I (beta(2)GPI) consists of five tandem repeated domains (I, II, III, IV, and V). The nicked form of beta(2)GPI (N-beta(2)GPI ) which was cleaved by plasmin in vitro at Lys 317-Thr 318 in domain V, showed reduced affinity for the negatively charged phospholipids, especially cardiolipin (CL). Recently, the N-beta(2)GPI was detected in the plasma of patients with disseminated intravascular coagulation syndrome (DIC) by an immunological method. In the present study, we prepared monoclonal antibodies for the nicked form, and demonstrated that the concentrations of this form of beta(2)GPI, which were analyzed by a sandwich ELISA using two specially prepared monoclonal antibodies, were significantly increased in the plasma of patients with leukemia (n = 51, mean +/- SD: 162.0 +/- 118.3 ng/ml) and with lupus anticoagulant (LA) (n =40, mean +/- SD: 3,041.5 +/- 16,579.7 ng/ml), compared to the normals (n = 33, mean +/- SD: 1.04 +/- 1.54 ng/ml). We found a significant correlation between the concentrations of N-beta(2)GPI and those of typical molecular markers for a fibrinolytic state such as plasmin-alpha(2) plasmin inhibitor complex (PIC) and D-dimer in patients with leukemia, but not in patients with LA. These results suggested that the generation of N-beta(2)GPI was caused by plasmin in the patients with leukemia, and by unknown proteases in the patients with LA. In the patients with LA, the levels of N-beta(2)GPI tended to be higher in those without thrombosis than in those with thrombosis.

Complementary DNA structure and genomic organization of Drosophila menin.

Menin is a protein product of a tumor supressor gene MEN1, mutations of which are responsible for multiple endocrine neoplasia type 1, an autosomal dominant familial cancer syndrome. We determined the nucleotide sequence of the Drosophila menin cDNA using RT-PCR and RACE, and confirmed it by direct sequencing of genomic DNA. Gene expression of Drosophila menin was detected by Northern blot analysis in adult and embryo as two types of transcripts, one identical in size to the cDNA, and the other larger but detected only in embryo. The Drosophila menin gene was composed of five exons in which the protein was encoded in exon 2 through 5, and spanned approximately 6.3 kb. The deduced amino acid (AA) sequence of Drosophila menin consisted of 751 AAs with a calculated molecular mass of 81.7 kDa, and showed 44-47% identity to human, rat, mouse and zebrafish menin over the entire length. Among the AA residue substitutions that have been reported as disease-associated missense mutations and single AA deletions, 53 out of 71 were completely conserved in Drosophila. The presence of menin ortholog in insect indicates that menin is an evolutionally conserved protein with a fundamental role in biological processes.

Early induction of the orphan nuclear receptor NOR-1 during cell death of the human breast cancer cell line MCF-7.

The neuron-derived orphan receptor (NOR-1) is a member of the NGFI-B subfamily within the nuclear receptor superfamily. In T-cell apoptosis, where NGFI-B plays an essential role, a functional redundancy between NGFI-B and NOR-1 has been demonstrated. Here, we examined the regulation and expression of the NOR-1 gene during cell death induced by a calcium ionophore A23187 in the human breast cancer cell line MCF-7. A23187 caused a transient increase in NOR-1 mRNA levels within 6 h after treatment. To delineate the sequences required for the transitional response to A23187, a series of promoter deletion mutants were constructed. From the transient transfection experiments, the element responsive to A23187 was identified between -94 and -42 base pairs upstream from the transcription initiation site. This 53-base pairs region contains three copies of the cAMP response element (CRE). Furthermore, phosphorylation of the CRE-binding protein (CREB), which affects the transcription of the CRE dependent-genes, was detected 30 min after A23187 stimulation. Our findings are consistent with NOR-1 involvement in A23187-induced cell death via the CRE-CREB signaling pathway.

Structure and distribution of rat menin mRNA.

Menin is a protein product of a tumor suppressor gene MEN1, mutations of which are responsible for multiple endocrine neoplasia type 1, an autosomal dominant familial cancer syndrome. We isolated rat menin cDNA clones from a fetal rat brain cDNA library. We also determined the nucleotide sequence of the protein coding region of mouse menin cDNA, which was partly registered in the expressed sequence tag (EST) database. Deduced amino acid sequences of rat and mouse menin are highly homologous to human menin. All of the previously reported disease-associated missense mutations and single amino acid deletions were observed at the residues that are conserved among these three species. Rat MEN1 transcripts were detected not only in the endocrine tissues but also in the tissues of the nervous, digestive, reproductive and immune systems. The MEN1 transcripts were abundantly expressed in the developing rat brain on day 14-18 of gestation. Immunoblotting and immunocytochemical analysis of the COS-7 cells transfected with a rat menin-expression vector revealed that the translated product has a molecular mass of approximately 70 kDa, and is localized mainly in the nucleus. These findings are consistent with those reported on human menin.