Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation

Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-β1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-β/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation.     

Effects of endogenous salicylic acid synthesized through PAL and ICS pathway on baicalin and baicalein accumulation in <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

The aim of this study was to investigate the effect of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS) on free salicylic acid (FSA) or total salicylic acid (TSA) content, and the effect of endogenous SA on baicalin and baicalein accumulation in Scutellaria baicalensis Georgi, respectively. We amplified partial sequences of PAL and ICS genes in Scutellaria baicalensis Georgi and silenced the two genes with virus-induced gene silence (VIGS) technique, respectively. The influence of gene silence on FSA, TSA, baicalin, and baicalein accumulation in Scutellaria baicalensis Georgi were analyzed, and these parameters were also investigated under high temperature. Results indicated that PAL silence significantly affected the FSA, ICS affected TSA content. FSA significantly affected the baicalin, rather than baicalein content. Our results along with previous studies indicated PAL and ICS were different in the regulation of FSA or TSA synthesis, and FSA and TSA were different in the regulation of baicalin and baicalein synthesis in Scutellaria baicalensis Georgi.     

Up-Regulation of microRNA-183 Promotes Cell Proliferation and Invasion in Glioma By Directly Targeting NEFL

Glioblastoma multiforme (GBM) is the most common and lethal type of primary malignant brain tumor. In recent years, increasing reports suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for human cancers, including GBM. The expression and roles of microRNA-183 (miR-183) has been explored in several types of human cancers, including in GBM, and plays important roles in tumor initiation and progression. However, its biological functions in GBM remain largely unknown. In this study, we demonstrated that miR-183 was significantly up-regulated in astrocytoma tissues and glioblastoma cell lines. Introduction of miR-183 mimics into U251 cells could promoted, while its antisense oligos inhibited cell proliferation and invasion. Moreover, we identified neurofilament light polypeptide (NEFL) as a novel target gene of miR-183. The expression levels of NEFL are inversely correlated with that of miR-183 in human astrocytoma clinical specimens. In addition, NEFL-siRNA could significantly attenuate the inhibitory effects of knockdown miR-183 on the proliferation and invasion of U251 cells via mTOR signaling pathway. Overall, This study revealed that miR-183 promotes glioma cell proliferation by targeting NEFL, and also demonstrated that miR-183 could be a potential target for GBM treatment.     

Growth and photosynthetic responses in Jatropha curcas L. seedlings of different provenances to watering regimes

Seedlings from four provenances of Jatropha curcas were subjected to 80, 50, and 30% of soil field capacity in potted experiments in order to study their responses to water availability. Our results showed that with the decline of soil water availability, plant growth, biomass accumulation, net photosynthetic rate, stomatal conductance (g_s), and transpiration rate (E) decreased, whereas leaf carbon isotope composition (δ¹³C), leaf pigment contents, and stomatal limitation value increased, while maximal quantum yield of PSII photochemistry was not affected. Our findings proved that stomatal limitation to photosynthesis dominated in J. curcas under low water availability. The increase of δ¹³C should be attributed to the decrease in g_s and E under the lowest water supply. J. curcas could adapt to low water availability by adjusting its plant size, stomata closure, reduction of E, increasing δ¹³C, and leaf pigment contents. Moreover, effects of provenance and the interaction with the watering regime were detected in growth and many physiological parameters. The provenance from xeric habitats showed stronger plasticity in the plant size than that from other provenances under drought. The variations may be used as criteria for variety/provenance selection and improvement of J. curcas performance.     

Novel <Emphasis Type="Italic">FBN1</Emphasis> mutations are responsible for cardiovascular manifestations of Marfan syndrome

The fibrillin-1 (FBN1) gene mutations result in Marfan syndrome (MFS) and have a variety of phenotypic variations. This disease is involved in the skeletal, ocular and cardiovascular system. Here we analyzed genotype-phenotype correlation in two Chinese families with MFS. Two patients with thoracic aortic aneurysms and dissections were diagnosed as MFS according to the revised Ghent criteria. Peripheral blood samples were collected and genomic DNAs were isolated from available cases, namely, patient-1 and his daughter and son, and patient-2 and his parents. According to the next-generation sequencing results, the mutations in FBN1 were confirmed by direct sequencing. A heterozygous frameshift mutation in exon 12 of FBN1 was found in the proband-1 and his daughter. They showed cardiovascular phenotype thoracic aortic aneurysms and dissections, a life-threatening vascular disease, and atrial septal defect respectively. One de novo missense mutation in exon 50 of FBN1 was identified only in the patient-2, showing aortic root aneurysm and aortic root dilatation. Intriguingly, two novel mutations mainly caused the cardiovascular complications in affected family members. No meaningful mutations were found in these two patients by screening all exons of 428 genes related with cardiovascular disease. The high incidence of cardiovascular manifestations might be associated with the two novel mutations in exon 12 and 50 of FBN1.     

A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting

To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K₂, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl₂ before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains.     

Insights into the solution structure of human deoxyhemoglobin in the absence and presence of an allosteric effector

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15N-1H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). To have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1H-15N J couplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed, and these changes are largely within the alpha1beta1 (or alpha2beta2) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.     

Solution structure of BmKalphaIT01, an alpha-insect toxin from the venom of the Chinese scorpion Buthus martensii Karsch

The solution structure of an alpha-insect toxin from Buthus martensii Karsch, BmKalphaIT01, has been determined by two-dimensional NMR spectroscopy and molecular modeling techniques. Combining the sequence homology comparison and toxicity bioassays, BmKalphaIT01 has been suggested to be a natural mutant of alpha-insect toxins and so can serve as a tool to study the relationship of structure-function among this group of toxins. The overall structure of BmKalphaIT01 shares a common core structure consisting of an alpha-helix packed against a three-stranded antiparallel beta-sheet, which exhibits distinctive local conformations within the loops connecting these secondary structure elements. The solution structure of BmKalphaIT01 features a non-proline cis peptide bond between Asn9 and Tyr10, which is proposed to mediate the spatial closing of the five-residue turn (Gln8-Cys12) and the C-terminal segment (Arg58-His64) to form the NC domain and confer the toxin insect-specific bioactivity. Conformational heterogeneity is observed in the solution of BmKalphaIT01 and could be attributed to the cis-trans isomerization of the peptide bond between residues 9 and 10. The minor conformation of BmKalphaIT01 with a trans peptide bond between Asn9 and Tyr10 may be responsible for its moderate bioactivity against mammals. The cis-trans isomerization of the peptide bond between residues 9 and 10 may be the structural basis of dual pharmacological activities of alpha-insect and alpha-like scorpion toxins, which is supported by the fact that conformational heterogeneity occurs in the solution structures of LqhalphaIT, LqqIII, and LqhIII and by comparison of the solution structure of BmKalphaIT01 with those of some relevant alpha-type toxins.     

Bombesin attenuates pre-mRNA splicing of glucocorticoid receptor by regulating the expression of serine-arginine protein p30c (SRp30c) in prostate cancer cells

Although glucocorticoids are frequently administered to patients with hormone refractory prostate cancer, their therapeutic effectiveness is limited by the development of glucocorticoid resistance. The molecular mechanisms of glucocorticoid resistance are unknown but are believed to involve neuropeptide growth factors and cytokines. We examined the functional interaction between bombesin and dexamethasone in PC-3 cells and found that bombesin could act as a survival factor by interfering with dexamethasone-mediated growth inhibition. Because glucocorticoids exert their effects through glucocorticoid receptors (GRs), we measured the expression of GR alpha and GR beta isoforms in the presence of bombesin. Western blotting and real time PCR revealed bombesin induced expression of GR beta, but not GR alpha. Because GR isoforms are generated by alternative splicing of a common GR gene, we examined the expression of serine-arginine (SR) proteins involved in alternative splicing, and found that the expression of SRp30 was induced by bombesin in PC-3 cells. To characterize the role of SRp30 in splicing of GR isoforms, siRNAs specific to various SRp30 isoforms were transfected into PC-3 cells. We found that suppression of SRp30c expression by siRNA specifically antagonized bombesin's effect on glucocorticoid-mediated inhibition of PC cells, suggesting that bombesin-induced expression of SRp30c affects GR pre-mRNA splicing, leading to increased GR beta expression and contributing to glucocorticoid resistance in PC cells.     

Identification of differentially expressed genes in omental adipose tissues of obese patients by suppression subtractive hybridization

To identify differentially expressed genes between obese individuals and normal control, we have undertaken suppression subtractive hybridization (SSH). Omental adipose tissues were obtained via abdominal surgery for appendicitis in both 13 obese subjects [BMI (body mass index) >30 kg/m2] and 13 normal subjects (BMI >18 and <25 kg/m2). Following SSH, about one thousand clones were sequenced and found to derive from 426 different genes. These predominately expressed genes included genes involved in lipid metabolism, cytokines, signal transduction, GLUT4 translocation, cell cycle and growth, cytoskeleton, and others. Although more detailed analyses are necessary, it is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of obesity.