Identification of a Novel Pathway of Transforming Growth Factor-β1 Regulation by Extracellular NAD+ in Mouse Macrophages: IN VITRO AND IN SILICO STUDIES

Extracellular β-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-β1.     

p27kip1 protein levels reflect a nexus of oncogenic signaling during cell transformation

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.     

A novel cardiomyocyte-enriched microRNA, miR-378, targets insulin-like growth factor 1 receptor: implications in postnatal cardiac remodeling and cell survival

Postnatal cardiac remodeling is characterized by a marked decrease in the insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R) expression. The underlying mechanism remains unexplored. This study examined the role of microRNAs in postnatal cardiac remodeling. By expression profiling, we observed a 10-fold increase in miR-378 expression in 1-week-old neonatal mouse hearts compared with 16-day-old fetal hearts. There was also a 4-6-fold induction in expression of miR-378 in older (10 months) compared with younger (1 month) hearts. Interestingly, tissue distribution analysis identified miR-378 to be highly abundant in heart and skeletal muscles. In the heart, specific expression was observed in cardiac myocytes, which was inducible by a variety of stressors. Overexpression of miR-378 enhanced apoptosis of cardiomyocytes by direct targeting of IGF1R and reduced signaling in Akt cascade. The inhibition of miR-378 by its anti-miR protected cardiomyocytes against H(2)O(2) and hypoxia reoxygenation-induced cell death by promoting IGF1R expression and downstream Akt signaling cascade. Additionally, our data show that miR-378 expression is inhibited by IGF1 in cardiomyocytes. In tissues such as fibroblasts and fetal hearts, where IGF1 levels are high, we found either absent or significantly low miR-378 levels, suggesting an inverse relationship between these two factors. Our study identifies miR-378 as a new cardioabundant microRNA that targets IGF1R. We also demonstrate the existence of a negative feedback loop between miR-378, IGF1R, and IGF1 that is associated with postnatal cardiac remodeling and with the regulation of cardiomyocyte survival during stress.     

In vitro metabolic stability of iodinated obestatin peptides

Different iodinated mouse obestatin peptides have been characterized toward their in vitro stability in the main metabolic compartments plasma, liver and kidney. Using HPLC-UV for quantification, significant differences in the degradation kinetics of the iodinated peptides, arising from both enzymatic proteolysis and dehalogenation, were found when compared to the native, unmodified peptide. HPLC-MS/MS analysis demonstrated that the cleavage sites were dependent upon the biological matrix and the location of the amino acid residue incorporating the iodine atom(s). The degrading proteases were found to target peptide bonds further away from the iodine incorporation, while proteolytic cleavages of nearby peptide bonds were more limited. Diiodinated amino acid residue containing peptides were found to be more susceptible to deiodination than the mono-iodinated derivative. In plasma, the percentage of peptide degradation solely attributed to deiodinase activity after 20 min incubation reached up to 25% for 2,5-diiodo-H(19)-obestatin compared to 20% and only 3% for (3,5-diiodo-Y(16))- and (3-iodo-Y(16)) obestatin, respectively. Hence, our results demonstrate that the different iodinated peptides pose significantly different metabolization properties and thus, also different biological activities are expected for peptides upon iodination.     

C-type Natriuretic Peptide: a novel biomarker of steroid induced bone toxicity in children with acute lymphoblastic leukemia (ALL)

Impaired bone growth and mineralization, and osteonecrosis are significant and common long-term sequelae of chemotherapy for childhood acute lymphoblastic leukemia (ALL). Here we have evaluated the relationship between linear bone growth during chemotherapy for ALL and bone derived C-type Natriuretic Peptide (CNP). CNP is known to be critical to normal endochondral bone growth in both rodents and humans, and plasma concentration of the amino terminal pro CNP (NTproCNP) is strongly correlated with concurrent height velocity in children. Plasma NTproCNP and CNP were measured by radio-immunoassay in 12 children aged 2-9 years during induction and maintenance chemotherapy for children with ALL. Height velocity was calculated from stadiometer readings at intervals of 3-12 months and related to plasma NTproCNP during each growth interval. Plasma NTproCNP was markedly suppressed in all subjects during induction chemotherapy. Brief periods of NTproCNP decline and rapid rebound during maintenance treatment coincided with the use of dexamethasone but not with other chemotherapeutics. Height velocity was markedly reduced during ALL induction but unaffected in maintenance phase, and these changes in growth were strongly correlated with plasma NTproCNP concentration. Plasma NTproCNP has potential use as a biomarker of glucocorticoid-induced bone toxicity.     

Antifungal properties of Canavalia ensiformis urease and derived peptides

Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO₂. These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli. The antifungal activity of ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H⁺ and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to ureases. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10⁻⁶–10⁻⁷ M.     

Genomic structure, polymorphism and expression analysis of the growth hormone (GH) gene in female and male Half-smooth tongue sole (Cynoglossus semilaevis)

Growth hormone (GH) is a polypeptide which is an important regulator of development and somatic growth in teleosts, and may be associated with the mechanisms which drive sexual growth dimorphism in the Half-smooth tongue sole (Cynoglossus semilaevis). In this study, the full length gh cDNA was cloned from C. semilaevis by homology cloning and the rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length gh cDNA is 826 bp and contains an open reading frame (ORF) of 603 bp encoding a protein of 200 amino acids (AA). The precursor of gh consists of a 17 amino-acid signal peptide followed by a 183 amino-acid mature polypeptide. GH gene sequences obtained from female and male adults consist of 3428 bp and 3371 bp, respectively, each of which includes six exons and five introns, and the difference in the GH gene size was mainly caused by the microsatellites. When 14 tissues from females, normal males and extra-large male adults were analyzed for sex-specific tissue expression, the gh mRNA was found to be predominantly expressed in the pituitary, and the expression levels in females were 3.6 times as much as those in normal males, while the mRNA expression in extra-large males was 1.7 times as much as those in normal males. Sex differences in gh mRNA expression during development were also examined by using a full-sib family of C. semilaevis, and the gh mRNA was detected at all of the 12 time points sampled from 10 to 380 days-old. A significant increase in gh mRNA was detected starting in 80 day old fish and was then followed by a drop to very low levels starting at 230 day old fish. Differential expression indicated that the gh expression level in females was significantly higher than males (P < 0.01) at all of the stages except for 10 days-old. Two microsatellite loci were identified in the second intron of the GH gene. Using these two polymorphic markers to genotype 224 individuals, there was no significant difference between the females and males in the Bohai Sea, the Yellow Sea and the hatchery samples.     

Depolymerised carrageenan enhances physiological activities and menthol production in Mentha arvensis L.

Irradiated carrageenan (IC) could elicit plant growth promoting activities in plants. The effect of foliar spray of five concentrations of IC (20, 40, 60_, 80 and 100 mg L⁻¹) was studied on Mentha arvensis L. in terms of plant growth, physiological attributes, herbage yield and the content and yield of essential oil and its components. Un-irradiated carrageenan and deionized water had no effect on the attributes studied. GPC study revealed formation of low molecular weight fractions in irradiated samples containing less than 20,000 molecular weight oligomers which are responsible for plant growth promotion in this study. 80 mg L⁻¹ of IC was the most effective concentration which resulted in the highest values of growth attributes, herbage yield and the content and yield of essential oil and menthol content of the oil. It also improved the leaf-nutrient contents, photosynthetic rate and other physiological parameters. 100 mg L⁻¹ of IC did not further improve the attributes studied, but it was always better than the control.     

Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.