Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro

The dental follicle (DF) surrounding the developing tooth germ is an ectomesenchymal tissue composed of various cell populations derived from the cranial neural crest. Human dental follicle cells (HDFC) are believed to contain precursor cells for cementoblasts, periodontal ligament cells, and osteoblasts. Bone morphogenetic proteins (BMPs) produced by Hertwig's epithelial root sheath or present in enamel matrix derivatives (EMD) seem to be involved in the control of DF cell differentiation, but their precise function remains largely unknown. We report the immunolocalization of STRO-1 (a marker of multipotential mesenchymal progenitor cells) and BMP receptors (BMPR) in DF in vivo. In culture, HDFC co-express STRO-1/BMPR and exhibit multilineage properties. Incubation with rhBMP-2 and rhBMP-7 or EMD for 24 h increases the expression of BMP-2 and BMP-7 by HDFC. Long-term stimulation of these cells by rhBMP-2 and/or rhBMP-7 or EMD significantly increases alkaline phosphatase activity (AP) and mineralization. Expression of cementum attachment protein (CAP) and cementum protein-23 (CP-23), two putative cementoblast markers, has been detected in EMD-stimulated whole DF and in cultured HDFC stimulated with EMD or BMP-2 and BMP-7. RhNoggin, a BMP antagonist, abolishes AP activity, mineralization, and CAP/CP-23 expression in HDFC cultures and the expression of BMP-2 and BMP-7 induced by EMD. Phosphorylation of Smad-1 and MAPK is stimulated by EMD or rhBMP-2. However, rhNoggin blocks only Smad-1 phosphorylation under these conditions. Thus, EMD may activate HDFC toward the cementoblastic phenotype, an effect mainly (but not exclusively) involving both exogenous and endogenous BMP-dependent pathways.     

Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24

Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP) located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.     

Structural Basis for Resistance to Diverse Classes of NAMPT Inhibitors

Inhibiting NAD biosynthesis by blocking the function of nicotinamide phosphoribosyl transferase (NAMPT) is an attractive therapeutic strategy for targeting tumor metabolism. However, the development of drug resistance commonly limits the efficacy of cancer therapeutics. This study identifies mutations in NAMPT that confer resistance to a novel NAMPT inhibitor, GNE-618, in cell culture and in vivo, thus demonstrating that the cytotoxicity of GNE-618 is on target. We determine the crystal structures of six NAMPT mutants in the apo form and in complex with various inhibitors and use cellular, biochemical and structural data to elucidate two resistance mechanisms. One is the surprising finding of allosteric modulation by mutation of residue Ser165, resulting in unwinding of an α-helix that binds the NAMPT substrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The other mechanism is orthosteric blocking of inhibitor binding by mutations of Gly217. Furthermore, by evaluating a panel of diverse small molecule inhibitors, we unravel inhibitor structure activity relationships on the mutant enzymes. These results provide valuable insights into the design of next generation NAMPT inhibitors that offer improved therapeutic potential by evading certain mechanisms of resistance.     

Agrobacterium-mediated genetic transformation of Withania somnifera using nodal explants

Withania somnifera is an important medicinal plant and used to cure many diseases. Direct regeneration method was standardized for the nodal explants of W. somnifera. In this method, the maximum of 42.4 ± 2.68 shoots produced per explant was achieved at 1.5 mg l^?1 BAP with 0.3 mg l^?1 IAA in the second subculture. Transformation was performed in the nodal explants of W. somnifera via direct regeneration using Agrobacterium tumefaciens strain EHA105 that harbored a binary vector pGA492, which carrying kanamycin resistant (nptII), phosphinothricin resistant (bar) and an intron containing β-glucuronidase (gus-intron) genes. The sensitivity of nodal explants to kanamycin (300 mg l^?1) was determined for the selection of transformed plants. Transformation was confirmed by histochemical β-glucuronidase (GUS) assay and amplification of the nptII gene by polymerase chain reaction (PCR). PCR and southern blot analyses confirmed the integration of nptII gene in the genome of W. somnifera and the transformation frequency of 3.16 % has been achieved. This is the first report on the genetic transformation of W. somnifera using nodal explants, which may aid in the transformation of any other character gene for improving therapeutic value.     

Experimental determination of sarcomere force–length relationship in type-I human skeletal muscle fibers

The objectives of this study were to measure the active and passive force–length (F–L) relationships in type-I human single muscle fibers and to compare the results to predictions from the sliding filament model (the “standard model”). We measured isometric forces in chemically skinned fibers at different sarcomere lengths (SLs) in separate maximal activations. The experimental tolerance interval for optimal SL was calculated to be (2.37, 2.95 μm), which included the prediction by the standard model (2.64, 2.81 μm). Average passive slack length was 2.22±0.08 μm, and the passive F–L relationship was well described by an exponential function. Best fit lines were used to estimate the ascending and descending limbs from the active F–L data using the average SL obtained from a digital image of the fiber. The experimental descending limb was also estimated using the shortest SL to address the possible effects of sarcomere inhomogeneity (SI). The experimental slopes of the ascending and descending limbs, 0.42 F_o/μm and ?0.52 F_o/μm (vs. ?0.55 F_o/μm with the shortest SL) respectively, F_o being the maximal isometric force, were significantly less in magnitude than those from the standard model. These results suggested that the difference between experimental and standard models was not fully explained by SI and other factors could be important. The broader experimental F–L curve compared to the standard model implies that human muscle has functionally a wider operating length range where its force-generating capacity is not compromised.     

Stearoyl-CoA desaturase-1 deficiency attenuates obesity and insulin resistance in leptin-resistant obese mice

Obesity and adiposity greatly increase the risk for secondary conditions such as insulin resistance. Mice deficient in the enzyme stearoyl-CoA desaturase-1 (SCD1) are lean and protected from diet-induced obesity and insulin resistance. In order to determine the effect of SCD1 deficiency on various mouse models of obesity, we introduced a global deletion of the Scd1 gene into leptin-deficient ob/ob mice, leptin-resistant Agouti (A^y/a) mice, and high-fat diet-fed obese (DIO) mice. SCD1 deficiency lowered body weight, adiposity, hepatic lipid accumulation, and hepatic lipogenic gene expression in all three mouse models. However, glucose tolerance, insulin, and leptin sensitivity were improved by SCD1 deficiency only in A^y/a and DIO mice, but not ob/ob mice. These data uncouple the effects of SCD1 deficiency on weight loss from those on insulin sensitivity and suggest a beneficial effect of SCD1 inhibition on insulin sensitivity in obese mice that express a functional leptin gene.     

Translational control of ceruloplasmin gene expression: beyond the IRE

Translational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor, erythroid 5-aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5'-untranslated region (UTR); and 15-lipoxygenase is regulated by specific proteins binding to the 3'-UTR. Ceruloplasmin (Cp) is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN)-gamma in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT), to a specific GAIT element in the Cp 3'-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.     

Modified methylenedioxyphenol analogs lower LDL cholesterol through induction of LDL receptor expression

Although statin therapy is a cornerstone of current low density lipoprotein (LDL)-lowering strategies, there is a need for additional therapies to incrementally lower plasma LDL cholesterol. In this study, we investigated the effect of several methylenedioxyphenol derivatives in regulating LDL cholesterol through induction of LDL receptor (LDLR). INV-403, a modified methylenedioxyphenol derivative, increased LDLR mRNA and protein expression in HepG2 cells in a dose- and time-dependent fashion. These effects were apparent even under conditions of HMG-CoA reductase inhibition. Electrophoresis migration shift assays demonstrated that INV-403 activates SREBP2 but not SREBP1c, with immunoblot analysis showing an increased expression of the mature form of SREBP2. Knockdown of SREBP2 reduced the effect of INV-403 on LDLR expression. The activation of SREBP2 by INV-403 is partly mediated by Akt/GSK3β pathways through inhibition of phosphorylation-dependent degradation by ubiquitin-proteosome pathway. Treatment of C57Bl/6j mice with INV-403 for two weeks increased hepatic SREBP2 levels (mature form) and upregulated LDLR with concomitant lowering of plasma LDL levels. Transient expression of a LDLR promoter-reporter construct, a SRE-mutant LDLR promoter construct, and a SRE-only construct in HepG2 cells revealed an effect predominantly through a SRE-dependent mechanism. INV-403 lowered plasma LDL cholesterol levels through LDLR upregulation. These results indicate a role for small molecule approaches other than statins for lowering LDL cholesterol.