Clinical features of SF3B1 mutation in patients with myelodysplastic syndrome with excess blasts / 中华内科杂志

Objective: To exploring the clinical features of SF3B1-mutated myelodysplastic syndrome with excess blasts (MDS-EB) and analyzing the association between SF3B1 mutation, and efficacy and prognostic significance for patients with MDS-EB. Methods: This was a retrospective case series study. The clinical data of 266 patients with MDS-EB diagnosed in the First Affiliated Hospital of Zhengzhou University between April 2016 and November 2021 were analyzed. The observed indicators included blood routine counts, mutated genes, overall response rate (ORR), overall survival (OS), progression-free survival (PFS), and leukemia-free survival (LFS). The Kaplan-Meier method was used to depict the survival curves. The Log-rank test method was equally used to compare survival across groups and performed the Cox proportional hazard regression model for prognostic analysis. Results: In 266 patients with MDS-EB, 166 (62.4%) were men, and the median age was 57 (17-81) years. Moreover, there were included 26 and 240 patients in the SF3B1-mutated and SF3B1 wild-type groups. Patients in the SF3B1-mutated group were older [median age 65 (51, 69) years vs. 56 (46, 66) years, P=0.033], had higher white blood cell (WBC) counts [3.08 (2.35, 4.78) × 109/L vs. 2.13 (1.40, 3.77) × 109/L], platelet (PLT) counts [122.5 (50.5, 215.0) ×109/L vs. 49.0 (24.3, 100.8) × 109/L], absolute neutrophil counts (ANC) [1.83 (1.01, 2.88) × 109/L vs. 0.80 (0.41, 1.99) × 109/L]and occurrence of DNMT3A mutation [23.1% (6/26) vs. 6.7% (16/240)] (all P<0.05). The ORR were similar in both groups after 2 and 4 cycles of therapy (P=0.348, P=1.000). Moreover, the LFS (P=0.218), PFS (P=0.179) and OS (P=0.188) were similar across the groups. Univariate Cox analysis revealed that SF3B1 mutation did not affect the prognosis of patients with MDS-EB (OS: P=0.193; PFS: P=0.184). Conclusions: Patients with SF3B1 mutation were older, with greater WBC, PLT, and ANC, and SF3B1 mutation easily co-occurred with DNMT3A mutation. From this model, there were no significant differences in efficacy and survival of MDS-EB with or without SF3B1 mutation.

Correlation between the mRNA levels of BCRP and LUNX genes and pathological types and stages of patients with non-small cell lung cancer / 中华医学遗传学杂志

OBJECTIVE@#To analyze the correlation between the mRNA levels of breast cancer resistance protein (BCRP) and lung-specific X protein (LUNX) genes with pathological types and stages of patients with non-small cell lung cancer (NSCLC) and their significance for prognosis.@*METHODS@#Eighty nine patients with NSCLC admitted to Huaihe Hospital of Henan University between June 2015 and June 2018 were recruited, with 55 patients with benign lung lesions admitted during the same period of time selected as the control group. The mRNA levels of BCRP and LUNX genes were detected in the peripheral blood samples from the two groups, and their correlation with the clinicopathological characteristics and prognosis of the patients was analyzed.@*RESULTS@#The expression rates of BCRP and LUNX mRNA in the NSCLC group were significantly higher compared with the control group (P < 0.05). The level of BCRP mRNA of the NSCLC patients has correlated with the degree of differentiation and TNM staging (P < 0.05), but not with gender, age, smoking, pathological types and lymph node metastasis (P > 0.05). The level of LUNX mRNA of them has correlated with the degree of differentiation, TNM staging and lymph node metastasis (P < 0.05), but not with gender, age, smoking, and pathological types (P > 0.05). Compared with those with no expression, the overall survival rate of patients with BCRP and LUNX expression was significantly lower (P < 0.05). The degree of differentiation, TNM staging, lymph node metastasis, and expression of the BCRP and LUNX mRNA may all affect the prognosis of the patients.@*CONCLUSION@#The levels of BCRP and LUNX mRNA in the peripheral blood of patients with NSCLC are significantly increased. The expression of BCRP mRNA is correlated with the degree of differentiation and TNM staging, whilst the expression of LUNX mRNA is correlated with the differentiation degree, TNM staging and lymph node metastasis. Both may be used as independent predictors for the prognosis of patients with NSCLC.

Analysis of a child with autosomal dominant mental retardation type 40 due to variant of CHAMP1 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical and genetic features of a child with autosomal dominant mental retardation type 40 (MRD40) due to variant of the CHAMP1 gene.@*METHODS@#Clinical characteristics of the child were analyzed. Genetic testing was carried out by low-depth high-throughput and whole genome copy number variant sequencing (CNV-seq) and whole exome sequencing (WES). A literature review was also carried out for the clinical phenotype and genetic characteristics of patients with MRD40 due to CHAMP1 gene variants.@*RESULTS@#The child, a 11-month-old girl, has presented with intellectual and motor developmental delay. CNV-seq revealed no definite pathogenic variants. WES has detected the presence of a heterozygous c.1908C>G (p.Y636*) variant in the CHAMP1 gene, which was carried by neither parent and predicted to be pathogenic. Literature review has identified 33 additional children from 12 previous reports. All children had presented with developmental delay and mental retardation, and most had dystonia (94.1%), delayed speech and/or walking (85.2%, 82.4%) and ocular abnormalities (79.4%). In total 26 variants of the CHAMP1 gene were detected, with all nonsense variants being of loss-of-function type, located in exon 3, and de novo in origin.@*CONCLUSION@#The heterozygous c.1908C>G (p.Y636*) variant of the CHAMP1 gene probably underlay the WRD40 in this child. Genetic testing should be considered for children featuring global developmental delay, mental retardation, hypertonia and facial dysmorphism.

Mutation of dentin sialophosphoprotein and hereditary malformations of dentin / 中华口腔医学杂志

The classification as well as the clinical manifestations of hereditary malformations of dentin are of great concern and have been deeply elucidated. The understanding of its genetic basis also increases progressively. Dentin sialophosphoprotein (DSPP) is the pathogenic gene of dentinogenesis imperfecta type Ⅱ, dentinogenesis imperfecta type Ⅲ and dentin dysplasia type Ⅱ. In this article, the classification of DSPP mutations as well as the resultant dysfunction of the mutant DSPP are summarized respectively and the corresponding clinical manifestations are analyzed. This work will provide a reference for the diagnosis and treatment of hereditary malformations of dentin.

Polymorphisms in DSSP (rs36094464) and RUNX2 (rs566712) genes contribute to the susceptibility of dental caries in childhood / Los polimorfismos en los genes DSSP (rs36094464) y RUNX2 (rs566712) contribuyen a la susceptibilidad de la caries dental en la infancia

SUMMARY: Dental caries corresponds to an ecological and non-contagious, dynamic and chronic disease of multifactorial origin; currently there is evidence of how genetic factors could be included as predisposing agents to suffer it, however this evidence is diverse and incipient. a cross-sectional study was p erformed to investigate the possible associations of DSPP (rs36094464), RUNX2 (rs566712) and KLK4 (rs198968) polymorphisms in early childhood caries. Saliva samples of children (2-11years old) were collected and genotyped for DSPP (rs36094464), RUNX2 (rs566712) and KLK4 (rs198968) polymorphisms. Through the ceft index their caries history was determined and the gene variants were students through molecular biology techniques. polymorphisms of the DSSP (rs36094464) and RUNX2 (rs566712) are associated and contribute to the susceptibility of dental caries disease in early childhood, as they are related to their history of caries. KLK4 (rs198968) polymorphisms are not associated. In conclusions, the studied polymorphisms on DSSP and RUNX2 genes are associated with changes in the tooth microarchitecture, favoring the appearance of microlesions that would contribute to dental caries disease susceptibility in early childhood. Also, no association was found for the studied polymorphism of the KLK4 gene with dental caries disease susceptibility.

RESUMEN: La caries dental corresponde a una enfermedad crónica, no contagiosa, dinámica y de origen multifactorial. Actualmente existe evidencia de cómo los factores genéticos podrían incluirse como agentes predisponentes, sin embargo, esta evidencia es diversa e incipiente. Se realizó un estudio transversal para investigar las posibles asociaciones entre los polimorfismos DSPP (rs36094464), RUNX2 (rs566712) y KLK4 (rs198968) y la caries en la infancia. Se colectaron muestras de saliva de niños (de 2 a 11 años de edad) y se genotipificaron para los polimorfismos DSPP (rs36094464), RUNX2 (rs566712) y KLK4 (rs198968). Mediante el índice ceft se determinó su historial de caries y se estudiaron las variantes genéticas mediante técnicas de biología molecular. Los datos obtenidos indican que los polimorfismos del DSSP (rs36094464) y RUNX2 (rs566712) están asociados y contribuyen a la susceptibilidad de la enfermedad de caries dental en la infancia, ya que están - además - relacionados con el historial de caries. En conclusión, los polimorfismos estudiados en los genes DSSP y RUNX2 se asocian a la aparición de microlesiones que contribuirían a la susceptibilidad a la enfermedad de caries dental en la infancia. Creemos que este estudio es importante para la odontopediatría porque destaca el papel de DSSP (rs36094464) y RUNX2 (rs566712) y la susceptibilidad a la caries dental durante la infancia, además resalta la utilidad de la evaluación genética para la predicción y prevención de la caries dental y porque aporta evidencia que indica que los factores genéticos están implicados en la etiología de la caries.

Isolation and characterization of a human cementocyte-like cell line, HCY-23

Abstract Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA

Abstract The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.

Comparison of Quantitation of Cytomegalovirus DNA by Real-Time PCR in Whole Blood with the Cytomegalovirus Antigenemia Assay

BACKGROUND: Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. METHOD: 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. RESULTS: The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. CONCLUSIONS: Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients.

Alteration of the SETBP1 Gene and Splicing Pathway Genes SF3B1, U2AF1, and SRSF2 in Childhood Acute Myeloid Leukemia

BACKGROUND: Recurrent somatic SET-binding protein 1 (SETBP1) and splicing pathway gene mutations have recently been found in atypical chronic myeloid leukemia and other hematologic malignancies. These mutations have been comprehensively analyzed in adult AML, but not in childhood AML. We investigated possible alteration of the SETBP1, splicing factor 3B subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine/arginine-rich splicing factor 2 (SRSF2) genes in childhood AML. METHODS: Cytogenetic and molecular analyses were performed to reveal chromosomal and genetic alterations. Sequence alterations in the SETBP1, SF3B1, U2AF1, and SRSF2 genes were examined by using direct sequencing in a cohort of 53 childhood AML patients. RESULTS: Childhood AML patients did not harbor any recurrent SETBP1 gene mutations, although our study did identify a synonymous mutation in one patient. None of the previously reported aberrations in the mutational hotspot of SF3B1, U2AF1, and SRSF2 were identified in any of the 53 patients. CONCLUSIONS: Alterations of the SETBP1 gene or SF3B1, U2AF1, and SRSF2 genes are not common genetic events in childhood AML, implying that the mutations are unlikely to exert a driver effect in myeloid leukemogenesis during childhood.

Distribution of non-LEE-encoded type 3 secretion system dependent effectors in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are important human gastroenteritis agents. The prevalence of six non-LEE genes encoding type 3 translocated effectors was investigated. The nleC, cif and nleB genes were more prevalent in typical than in atypical EPEC, although a higher diversity of genes combinations was observed in atypical EPEC.

Role of LPA and the Hippo pathway on apoptosis in salivary gland epithelial cells

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid involved in numerous physiological responses. However, the expression of LPA receptors and the role of the Hippo signaling pathway in epithelial cells have remained elusive. In this experiment, we studied the functional expression of LPA receptors and the associated signaling pathway using reverse transcriptase-PCR, microspectrofluorimetry, western blotting and immunocytochemistry in salivary gland epithelial cells. We found that LPA receptors are functionally expressed and involved in activating the Hippo pathway mediated by YAP/TAZ through Lats/Mob1 and RhoA/ROCK. Upregulation of YAP/TAZ-dependent target genes, including CTGF, ANKRD1 and CYR61, has also been observed in LPA-treated cells. In addition, based on data suggesting that tumor necrosis factor (TNF)-alpha induces cell apoptosis, LPA upregulates TNF-induced caspase-3 and cleaved Poly(ADP-ribose)polymerase (PARP). However, small interfering RNA treatment to Yes-associated protein (YAP) or transcriptional co-activator with a PDZ-binding motif (TAZ) significantly decreased TNF-alpha- and LPA-induced apoptosis, suggesting that YAP and TAZ modulate the apoptotic pathway in salivary epithelial cells.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Nueva mutación heterocigota en el gen de la proteína regulatoria aguda de la esteroideogénesis (StAR) en un paciente 46,XY con hiperplasia adrenal congénita lipoidea / [Novel heterozygous mutation in the steroidogenic acute regulatory protein gene in a 46,XY patient with congenital lipoid adrenal hyperplasia].

StAR facilitates cholesterol entry into the mitochondria as part of the transduceosome complex. Recessive mutations in the gen STAR cause classic and nonclassic congenital lipoid adrenal hyperplasia. The aim of the study was to analyze the molecular consequences of a novel heterozygous STAR mutation in a 46,XY patient with ambiguous genitalia and adrenal insufficiency. We found a de novo heterozygous IVS-2A>G STAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1 and TSPO genes. RT-PCR and sequencing from patient’s testicular RNA showed a -exon2 transcript and the wild-type (WT) transcript. Both 37 kDa precursor and 30 kDa mature protein were detected in COS-7 cell transfected with mutant and WT plasmids. Immunofluorescence showed almost no co-localization of mitochondria and mutant protein (delta22-59StAR). Delta22-59StAR activity was 65±13

of WT. Cotransfection with WT and delta22-59StAR plasmids reduced WT activity by 62.0

± 13.9. Novel splice-junction heterozygous STAR mutation (IVS-2A>G) resulted in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. A misfolded p.G22_L59delStAR might interfere with WT StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype.

Cloning and expression analysis of multiple proteins encoding P gene of Newcastle disease virus.

Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.

Clinical Features of Congenital Adrenal Insufficiency Including Growth Patterns and Significance of ACTH Stimulation Test

Congenital adrenal insufficiency is caused by specific genetic mutations. Early suspicion and definite diagnosis are crucial because the disease can precipitate a life-threatening hypovolemic shock without prompt treatment. This study was designed to understand the clinical manifestations including growth patterns and to find the usefulness of ACTH stimulation test. Sixteen patients with confirmed genotyping were subdivided into three groups according to the genetic study results: congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH, n=11), congenital lipoid adrenal hyperplasia (n=3) and X-linked adrenal hypoplasia congenita (n=2). Bone age advancement was prominent in patients with CAH especially after 60 months of chronologic age (n=6, 67%). They were diagnosed in older ages in group with bone age advancement (P<0.05). Comorbid conditions such as obesity, mental retardation, and central precocious puberty were also prominent in this group. In conclusion, this study showed the importance of understanding the clinical symptoms as well as genetic analysis for early diagnosis and management of congenital adrenal insufficiency. ACTH stimulation test played an important role to support the diagnosis and serum 17-hydroxyprogesterone levels were significantly elevated in all of the CAH patients. The test will be important for monitoring growth and puberty during follow up of patients with congenital adrenal insufficiency.

Disruption of reproductive development in male rat offspring following gestational and lactational exposure to di-(2-ethylhexyl) phthalate and genistein

Studies of developmental effects of mixtures of endocrine disrupters on the male reproductive system are of great concern. In this study, the reproductive effects of the co-administration of di-2-(ethylhexyl) phthalate (DEHP) and genistein (GEN) during pregnancy and lactation were studied in male rat offspring. Pregnant Sprague-Dawley rats were gavaged from gestation day 3 to postnatal day 21 with vehicle control, DEHP 250 mg/kg body weight (bwyday, GEN 50 mg/kg bwday, GEN 400 mg/kg bwday, and two combinations of the two compounds (DEHP 250 mg/kg bwday + GEN 50 mg/kg bwday, DEHP 250 mg/kg bwday + GEN 400 mg/kg bwday). The outcomes studied were general morphometry (weight, AGD), testicular histology, testosterone levels, and expression at the mRNA level of genes involved in steroidogenesis. Organ coefficient, AGD / body weight1/3 י, serum testosterone concentration and genes involved in steroidogenic pathway expression when exposed to DEHP (250mg/kg bwday), GEN(50mg/kg bwday) or GEN(400mg/kg bwday) alone were not significantly different from the control group. When exposed to (DEHP 250mg/kg bwday +GEN 50mg/kg bwday) together during pregnancy and lactation, serum testosterone concentration, epididymis coefficient and Cypal17a1,Scarb1 m RNA expression significantly decreased compared to the control and GEN(50mg/kg bwday). When exposed to (DEHP 250mg/kg bwday +GEN 400mg/kg bwday) together during pregnancy and lactation, AGD / body weight1/3 י, serum testosterone concentration, testis and epididymis coefficient and Star, Cypal17a1 mRNA expression appeared significantly decreased compared to the control and DEHP/GEN single exposure, together with developmental impairment of seminiferous tubules and seminiferous epithelium. Overall, co-administration of DEHP and GEN during gestation and lactation seem to acts in a cumulative manner to induce the most significant alterations in the neonate, especially with GEN at high dose, although the effect of the DEHP-GEN mixture on adult offspring should be observed further.

Antiobesity activity of a sphingosine 1-phosphate analogue FTY720 observed in adipocytes and obese mouse model

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.

Dendritic eIF4E-binding Protein 1 (eIF4E-BP1) mRNA Is Upregulated by Neuronal Activation

Long-term synaptic plasticity requires addition of new proteins at the synaptic site. The local protein synthesis at subsynaptic sites confers advantageous mechanisms that would regulate the protein composition in local domains on a moment-by-moment basis. However, our information on the identities of 'dendritic' mRNAs is very limited. In this study we investigated the expression of the protein and mRNA for eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) in cultured rat hippocampal neurons. Immunocytochemistry (ICC) showed that 4EBP1 protein is highly localized to the nucleus. In dendrites most 4EBP1 punctae were not colocalized with those of eIF4E. In situ hybridization (ISH) and Fluorescence ISH (FISH) revealed that 4EBP1 mRNA was present in dendrites. The FISH signals formed clusters along dendrites that colocalized with ICC signals for Staufen, a marker for RNA granules. The neuronal activation by KCl (60 mM, 10 min) significantly increased the density of 4EBP1 FISH signals in the nucleus after 2 hr, and both in the nucleus and dendrites after 6 hr. Our results indicate that 4EBP1 and its mRNA are present in dendrites, and the mRNA is upregulated and transported to dendritic domains in RNA granules upon neuronal activation.

Negative feedback regulation of Wnt signaling by Gbetagamma-mediated reduction of Dishevelled

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.