Difluoromethylornithine stimulates early cardiac commitment of mesenchymal stem cells in a model of mixed culture with cardiomyocytes

The efficiency of in vitro mesenchymal stem cell (MSC) differentiation into the myocardial lineage is generally poor. In order to improve cardiac commitment, bone marrow GFP+MSCs obtained from transgenic rats were cultured with adult wild type rat cardiomyocytes for 5 days in the presence of difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis and cell proliferation. The percentage of GFP+MSCs showing cardiac myofibril proteins (cMLC2, cTnI) was about threefold higher after DFMO addition (3%) relative to the untreated control (1%). Another set of experiments was performed with cardiomyocytes incubated for 1 day in the absence of glucose and serum and under hypoxic conditions (pO2 < 1%), in order to simulate severe ischemia. The percentage of cardiac committed GFP+MSCs was about 5% when cultured with the hypoxic/starved cardiomyocytes and further increased to 7% after DFMO addition. The contemporary presence of putrescine in DFMO-treated cells markedly blunted differentiation, while the cytostatic mitomycin C was not able to induce cardiac commitment. The involvement of histone acetylation in DFMO-induced differentiation was evidenced by the strong attenuation of cardiac commitment exerted by anacardic acid, an inhibitor of histone acetylase. Moreover, the percentage of acetylated histone H3 significantly increased in bone marrow MSCs obtained from wild type rats and treated with DFMO. These results suggest that polyamine depletion can represent a useful strategy to improve MSC differentiation into the cardiac lineage, especially in the presence of cardiomyocytes damaged by an ischemic environment.     

Comparative proteomics lends insight into genotype‐specific pathogenicity

Comparative proteomic analyses have emerged as a powerful tool for the identification of unique biomarkers and mechanisms of pathogenesis. In this issue of Proteomics, Murugaiyan et al. utilize difference gel electrophoresis (DIGE) to examine differential protein expression between nonpathogenic and pathogenic genotypes of Prototheca zopfii, a causative agent in bovine enteritis and mastitis. Their findings provide insights into molecular mechanisms of infection and evolutionary adaptation of pathogenic genotypes, demonstrating the power of comparative proteomic analyses.     

Evolution at the tip and base of the X chromosome in an African population of Drosophila melanogaster

Hitchhiking effects of advantageous mutations have been invoked to explain reduced polymorphism in regions of low crossing-over in Drosophila. Besides reducing DNA heterozygosity, hitchhiking effects should produce strong linkage disequilibrium and a frequency spectrum skewed toward an excess of rare polymorphisms (compared to the neutral expectation). We measured DNA polymorphism in a Zimbabwe population of D. melanogaster at three loci, yellow, achaete, and suppressor of forked, located in regions of reduced crossing-over. Similar to previously published surveys of these genomic regions in other populations, we observed low levels of nucleotide variability. However, the frequency spectrum was compatible with a neutral model, and there was abundant evidence for recombination in the history of the yellow and ac genes. Thus, some aspects of the data cannot be accounted for by a simple hitchhiking model. An alternative hypothesis, background selection, might be compatible with the observed patterns of linkage disequilibrium and the frequency spectrum. However, this model cannot account for the observed reduction in nucleotide heterozygosity. Thus, there is currently no satisfactory theoretical model for the data from the tip and base of the X chromosome in D. melanogaster.      

Short chain oligogalacturonides induce ethylene production and expression of the gene encoding aminocyclopropane 1-carboxylic acid oxidase in tomato plants

Oligogalacturonic acids (OGAs), derived from plant cell wall pectin, have been implicated in a number of signal transduction pathways involved in growth, development and defense responses of higher plants. This study investigates the size range of OGAs capable of inducing ethylene synthesis in tomato plants, and demonstrates that in contrast with many other effects, only short chain OGAs are active. Oligomers across a range of DP from 2-15 were separated and purified to homogeneity by QAE-Sephadex anion exchange chromatography using a novel elution system. The OGAs were applied to tomato plants and assayed for their ability to induce ethylene gas release and changes in steady state levels of mRNA encoding the ethylene forming enzyme aminocyclopropane-1-carboxylic acid oxidase (ACO). The study demonstrated that only OGAs in the size range of DP4-6 were active both in eliciting ACO expression and in the production of ethylene.      

Evaluation of home-based thermal biofeedback treatment of pediatric migraine headache

Sixteen children and adolescents with migraine headache were treated with thermal biofeedback. Seven were seen individually in the clinic while the other nine participated in a limited-contact, partly home-based regimen. Evaluation of headache diary data from 4-week monitoring periods before and after treatment showed significant (p<.01) reduction in headaches, with no significant difference in efficacy between the two conditions.This research was supported in part by grants from NIMH, MH-41341, and NINCDS, NS-23440.     

Opioid receptors in rat cardiac sarcolemma: effect of phenylephrine and isoproterenol

The present study demonstrates the presence of opioid receptors in the rat cardiac sarcolemma isolated by the hypotonic LiBr-shock procedure. Opioid binding was measured by using [3H]U69 593, [3H](2-D-penicillamine,5-D-penicillamine)enkephalin ([3H]DPDPE) or [3H][D-Ala2,MePhe4,Gly-(ol)5]enkephalin ([3H]DAGO) as selective radioligands for K, delta and mu opioid receptors, respectively. Both the K- and delta-selective ligands exhibited highly specific (75-86%) binding, saturable at a concentration of about 20 nM. No specific binding for the selective agonist DAGO was observed. A marked increase in both [3H]U69 593 and [3H]DPDPE binding was observed after incubation of the sarcolemma with the alpha-adrenoceptor agonist phenylephrine or with the beta-adrenoceptor agonist isoproterenol. These stimulatory effects were associated with an increase in the Bmax values, a decrease in the Kd values, and were completely antagonized by the respective antagonists phentolamine and propranolol.     

Activation of the cannabinoid CB1 receptor may involve a W6 48/F3 36 rotamer toggle switch.

The cannabinoid CB1 receptor, a member of the Rhodopsin (Rho) family of G protein coupled receptors (GPCRs), exhibits high levels of constitutive activity. In contrast, Rho exhibits an exquisite lack of constitutive activity. In Rho, W6.48(265) on transmembrane helix 6 (TMH6) is flanked by aromatic residues at positions i-4 (F6.44) and i + 3 (Y6.51), while in CB1 the residues i-4 and i + 3 to W6.48 are leucines (L6.44 and L6.51). Based upon spectroscopic evidence, W6.48 has been proposed to undergo a rotamer switch (chi1 g+ -->trans) upon activation of Rho. In the work reported here, the biased Monte Carlo method, Conformational Memories (CM) was used to test the hypothesis that the high constitutive activity exhibited by CB1 may be due, in part, to the lack of aromatic residues i-4 and i + 3 from W6.48. In this work, the W6.48 rotamer shift (chi1 g+ -->trans) was used as the criterion for activation. Conformational Memories (CM) calculations on WT CB1 TMH6 and L6.44F and L6.51Y mutant TMH6s revealed that an aromatic residue at 6.44 tends to disfavor the W6.48 chi1 g+ -->trans transition and an aromatic residue at 6.51 would require a concomitant movement of the Y6.51 chi1 from trans-->g+ when the W6.48 chi1 undergoes a g+ -->trans shift. In contrast, CM calculations on WT CB1 TMH6 revealed that the presence of leucines at 6.44 and 6.51 provide W6.48 with greater conformational mobility, with a W6.48 transchi1 preferred. Conformational Memories calculations also revealed that the W6.48 chi1 g+ -->trans transition in WT CB1 TMH6 is correlated with the degree of kinking in TMH6. The average proline kink angles for TMH6 were higher for helices with a W6.48 g+ chi1 than for those with a W6.48 transchi1. These results are consistent with experimental evidence that TMH6 straightens during activation. Transmembrane helix (TMH) bundle models of the inactive (R) and active (R*) states of CB1 were then probed for interactions that may constrain W6.48 in the inactive state of CB1. These studies revealed that F3.36 (transchi1) helps to constrain W6.48 in a g+ chi1 in the inactive (R) state of CB1. In the R* state, these studies suggest that F3.36 must assume a g+ chi1 in order to allow W6.48 to shift to a transchi1. These results suggest that the W6.48/F3.36 interaction may act as the 'toggle switch' for CB1 activation, with W6.48 chi1 g+/F3.36 chi1 trans representing the inactive (R) and W6.48 chi1 trans/F3.36 chi1 g+ representing the active (R*) state of CB1.     

Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart

Cardiac ischemia may be responsible for either the loss of endothelial nitric oxide synthase (eNOS) or changes in its activity, both conditions leading to coronary dysfunction. We investigated whether early ischemic preconditioning was able to preserve eNOS protein expression and function in the ischemic/reperfused myocardium. Langendorff-perfused rat hearts were subjected to 20 min global ischemia, followed by 30 min reperfusion (I/R). A second group of hearts was treated as I/R, but preconditioned with three cycles of 5 min-ischemia/5 min-reperfusion (IP). Cardiac contractility markedly decreased in I/R, consistently with the rise of creatine kinase (CK) activity in the coronary effluent, whilst ischemic preconditioning significantly improved all functional parameters and reduced the release of CK. Western blot analysis revealed that the amount of eNOS protein decreased by 54.2% in I/R with respect to control (p < 0.01). On the other hand, NOS activity was not significantly reduced in I/R, as well as cGMP tissue levels, suggesting that a parallel compensatory stimulation of this enzymatic activity occurred during ischemia/reperfusion. Ischemic preconditioning completely prevented the loss of eNOS. Moreover, both NOS activity and cGMP tissue level were significantly higher (p < 0.05) in IP (12.7 +/- 0.93 pmol/min/mg prot and 58.1 +/- 12.2 fmol/mg prot, respectively) than I/R (7.34 +/- 2.01 pmol/min/mg prot and 21.4 +/- 4.13 fmol/mg prot, respectively). This suggest that early ischemic preconditioning may be useful to accelerate the complete recovery of endothelial function by preserving the level of cardiac eNOS and stimulating the basal production of nitric oxide.     

Chromosome imbalances in oligodendroglial tumors detected by comparative genomic hybridization

Seven well-differentiated oligodendrogliomas, 16 anaplastic oligodendrogliomas and two cases of oligoastrocytomas were investigated by comparative genomic hybridization (CGH) on frozen tissue samples. The most frequent losses found involved 1p and 19q in 32% of cases. Loss of 9p was observed during malignant progression in 25% of anaplastic oligodendrogliomas. In two anaplastic oligodendrogliomas gain of 1q was found. The frequent losses of chromosome 16 and 22 have not been reported previously. These results underscore that CGH is a powerful tool for the classification of gliomas complementing the traditional histopathological approach.     

Modulation of ornithine decarboxylase activity and ornithine decarboxylase-antizyme complex in rat heart by hormone and putrescine treatment

Ornithine decarboxylase was present in a cryptic, complexed form in an amount approximately equivalent to that of free ornithine decarboxylase activity in adult rat heart. Addition of isoproterenol (10 mg/kg) caused a notable rise in ornithine decarboxylase activity and a simultaneous decrease in the amount of the complexed enzyme. During the period of ornithine decarboxylase decay, when cardiac putrescine content had reached high values, the level of the complex increased above that of the control. Administration of putrescine (1.5 mmol/kg, twice) or dexamethasone (4 mg/kg) produced a decrease of heart ornithine decarboxylase activity, while it did not remarkably affect the level of complexed ornithine decarboxylase, therefore raising significantly the ratio of bound to total ornithine decarboxylase. Putrescine also elicited the appearance of free antizyme, concomitantly with the disappearance of free ornithine decarboxylase activity after 3-4 h of treatment. These results indicate that a significant amount of ornithine decarboxylase occurs in an inactive form in the heart under physiological conditions and that its absolute and relative levels may vary following stimuli which affect heart ornithine decarboxylase activity.