The role of mechanical forces in dextral rotation during cardiac looping in the chick embryo

Cardiac looping is a vital morphogenetic process that transforms the initially straight heart tube into a curved tube normally directed toward the right side of the embryo. While recent work has brought major advances in our understanding of the genetic and molecular pathways involved in looping, the biophysical mechanisms that drive this process have remained poorly understood. This paper examines the role of biomechanical forces in cardiac rotation during the initial stages of looping, when the heart bends and rotates into a c-shaped tube (c-looping). Embryonic chick hearts were subjected to mechanical and chemical perturbations, and tissue stress and strain were studied using dissection and fluorescent labeling, respectively. The results suggest that (1) the heart contains little or no intrinsic ability to rotate, as external forces exerted by the splanchnopleure (SPL) and the omphalomesenteric veins (OVs) drive rotation; (2) unbalanced forces in the omphalomesenteric veins play a role in left-right looping directionality; and (3) in addition to ventral bending and rightward rotation, the heart tube also bends slightly toward the right. The results of this study may help investigators searching for the link between gene expression and the mechanical processes that drive looping.     

Monastrol, a prototype anti-cancer drug that inhibits a mitotic kinesin, induces rapid bursts of axonal outgrowth from cultured postmitotic neurons

Terminally postmitotic neurons continue to express many of the kinesin-related proteins known to configure microtubules during mitosis. Drugs that inhibit these kinesins are being developed as anti-cancer agents with the hope that they will inhibit proliferation of tumor cells without having adverse effects on the nervous system. The prototype, termed monastrol, inhibits the kinesin known as Eg5, which is essential for maintaining separation of the half-spindles. Eg5 is also highly expressed in neurons, particularly during development. Exposure of cultured sympathetic neurons to monastrol for a few hours increased both the number and the growth rate of the axons. With additional time, the overall lengths of the axons were indistinguishable from controls. Sensory neurons showed a similar short-term increase in axonal growth rate. However, prolonged exposure resulted in shorter axons, suggesting that sensory neurons may be more sensitive to toxic effects of the drug. Nevertheless, the overall health of the cultures was still far more robust than cultures treated with taxol, a drug commonly used for anti-cancer therapy. On the basis of these results, we conclude that Eg5 normally generates forces that oppose axonal growth, presumably by partially suppressing the forward advance of microtubules. We speculate that local regulation of Eg5 could be a means by which neurons coordinate rapid bursts of axonal growth with appropriate environmental cues. The comparatively modest toxic effects on the neurons over time are a hopeful sign for clinicians interested in using anti-Eg5 drugs for cancer therapy.     

Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein

Elongation of very long chain fatty acids 4 (ELOVL4) is a novel member of the ELO family of genes that are involved in fatty acid metabolism. ELOVL4 encodes a putative transmembrane protein of 314 amino acids that carries a possible endoplasmic reticulum (ER) retention/retrieval signal (KXKXX) at the C-terminus. Two distinct mutations, a 5-bp deletion and a complex mutation from the same region in exon 6 of this gene, have been reported so far and are associated with autosomal dominant atrophic macular degeneration (adMD/STGD3). Both of these deletions could result in C-terminal truncation and loss of the ER retention signal in the mutant protein. We expressed the wild-type and mutant proteins in COS-7 and CHO cells to study the intracellular distribution of ELOVL4 and to identify possible implications of the above mutations in its localization. Immunofluorescence analysis of these proteins along with organelle marker antibodies revealed predominant ER localization for wild-type ELOVL4. Targeted deletion of the dilysine motif at the C-terminus of the protein resulted in the loss of ER localization. Immunoelectron microscopy and immunofluorescence analysis revealed a similar ER localization pattern for the protein in human photoreceptors. These data indicate that ELOVL4 is an ER-resident protein, which supports its suggested function in fatty acid elongation. We also demonstrate that the localization of both mutant proteins was dramatically changed from an ER to a Golgi distribution. Our observations suggest that the consequences of defective protein trafficking could underlie the molecular mechanism associated with degeneration of the macula in the patients with adMD/STGD3.     

The cytotoxic activity of lactoperoxidase: enhancement and inhibition by neuroactive compounds

Neuronal death associated with Parkinson's disease is commonly believed to be caused by oxygen- and nitrogen-derived free radical species. Some years ago, however, we showed that peroxidase from the midbrain of dogs is able to kill various cell types, including neuroblastoma cells (M. B. Grisham et al., J. Neurochem. 48: 876-882: 1987). We postulated that a nigral peroxidase may play a significant role in the degeneration of dopaminergic neurons in Parkinson's disease. To further establish proof of principle, we recently performed a series of experiments using horseradish peroxidase and lactoperoxidase. We showed that the cytotoxic activity of lactoperoxidase is fully inhibited by physiological concentrations of dopamine, reduced glutathione, and L-cysteine, as well as by micromolar concentrations of apomorphine, desferal, aspirin, and uric acid. l-Methyl-4-phenyl-1,2-dihydropyridine (MPDP) and l-methyl-4-phenylpyridinium (MPP+) augment the cytotoxic activity, whereas l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, deprenyl, and pargyline had minimal or no effect. We also showed that horseradish peroxidase catalyzes the oxidation of MPDP to MPP+. Thus, contrary to the generally accepted theory that the in vivo oxidation of MPDP occurs spontaneously, this reaction may be catalyzed by a brain peroxidase. These observations lend further support to the suggestion that a brain peroxidase may play an important role in the metabolic events associated with Parkinson's disease.     

Activator of G protein signaling 3: a gatekeeper of cocaine sensitization and drug seeking

Chronic cocaine administration reduces G protein signaling efficacy. Here, we report that the expression of AGS3, which binds to GialphaGDP and inhibits GDP dissociation, was upregulated in the prefrontal cortex (PFC) during late withdrawal from repeated cocaine administration. Increased AGS3 was mimicked in the PFC of drug-naive rats by microinjecting a peptide containing the Gialpha binding domain (GPR) of AGS3 fused to the cell permeability domain of HIV-Tat. Infusion of Tat-GPR mimicked the phenotype of chronic cocaine-treated rats by manifesting sensitized locomotor behavior and drug seeking and by increasing glutamate transmission in nucleus accumbens. By preventing cocaine withdrawal-induced AGS3 expression with antisense oligonucleotides, signaling through Gialpha was normalized, and both cocaine-induced relapse to drug seeking and locomotor sensitization were prevented. When antisense oligonucleotide infusion was discontinued, drug seeking and sensitization were restored. It is proposed that AGS3 gates the expression of cocaine-induced plasticity by regulating G protein signaling in the PFC.     

Activity in posterior parietal cortex is correlated with the relative subjective desirability of action

Behavioral studies suggest that making a decision involves representing the overall desirability of all available actions and then selecting that action that is most desirable. Physiological studies have proposed that neurons in the parietal cortex play a role in selecting movements for execution. To test the hypothesis that these parietal neurons encode the subjective desirability of making particular movements, we exploited Nash's game theoretic equilibrium, during which the subjective desirability of multiple actions should be equal for human players. Behavior measured during a strategic game suggests that monkeys' choices, like those of humans, are guided by subjective desirability. Under these conditions, activity in the parietal cortex was correlated with the relative subjective desirability of actions irrespective of the specific combination of reward magnitude, reward probability, and response probability associated with each action. These observations may help place many recent findings regarding the posterior parietal cortex into a common conceptual framework.     

Homer proteins regulate sensitivity to cocaine

Drug addiction involves complex interactions between pharmacology and learning in genetically susceptible individuals. Members of the Homer gene family are regulated by acute and chronic cocaine administration. Here, we report that deletion of Homer1 or Homer2 in mice caused the same increase in sensitivity to cocaine-induced locomotion, conditioned reward, and augmented extracellular glutamate in nucleus accumbens as that elicited by withdrawal from repeated cocaine administration. Moreover, adeno-associated virus-mediated restoration of Homer2 in the accumbens of Homer2 KO mice reversed the cocaine-sensitized phenotype. Further analysis of Homer2 KO mice revealed extensive additional behavioral and neurochemical similarities to cocaine-sensitized animals, including accelerated acquisition of cocaine self-administration and altered regulation of glutamate by metabotropic glutamate receptors and cystine/glutamate exchange. These data show that Homer deletion mimics the behavioral and neurochemical phenotype produced by repeated cocaine administration and implicate Homer in regulating addiction to cocaine.     

Enhanced IGF-1 expression improves smooth muscle cell engraftment after cell transplantation

The functional benefit of cell transplantation after a myocardial infarction is diminished by early cell losses. IGF-1 enhances cell proliferation and survival. We hypothesized that IGF-1-transfected smooth muscle cells (SMCs) would enhance cell survival and improve engraftment after cell transplantation. The IGF-1 gene was transfected into male SMCs and compared with SMCs transfected with a plasmid vector (vector control) and nontransfected SMCs (cell control). IGF-1 mRNA (n=10/group) and protein levels (n=6/group) were higher (P <0.05 for all groups) at 3, 7, and 14 days compared with controls. VEGF was also increased in parallel to enhanced IGF-1 expression. IGF-1-transfected cells demonstrated greater cell proliferation, stimulated angiogenesis, and decreased caspase-3 activity after simulated ischemia and reperfusion (P <0.05 for all groups compared with vector or cell controls). A uniform left ventricular injury was produced in female rats using a cryoprobe. Three weeks later, 2 x 10(6) cells from three groups were implanted into the scar. One week later, IGF-1-transfected SMCs had increased myocardial IGF-1 and VEGF levels, increased Bcl2 expression, limited cell apoptosis, and enhanced vessel formation in the myocardial scar compared with the two control groups (P <0.05 for all groups). The proportion of SMCs surviving in the implanted region was greater (P <0.05) in the IGF-1-transfected group than in the vector or cell controls. Gene enhancement with IGF-1 improved donor cell proliferation, survival, and engraftment after cell transplantation, perhaps mediated by enhanced angiogenesis and reduced apoptosis.     

Genetic structuring of Patagonian toothfish populations in the Southwest Atlantic Ocean: the effect of the Antarctic Polar Front and deep-water troughs as barriers to genetic exchange

The environmental and/or life history factors affecting genetic exchange in marine species with potential for high dispersal are of great interest, not only from an evolutionary standpoint but also with regard to effective management. Previous genetic studies have demonstrated substantial differentiation among populations of the Patagonian toothfish around the Southern Ocean, indicating breakdown of gene flow across large distances between inhabited shelf areas. The present study examined genetic structuring through analysis of microsatellite loci and restriction fragment length polymorphism (RFLP) of the mitochondrial ND2 gene and control region of the toothfish population in the SW Atlantic, allowing examination of the relative effects of the Antarctic Polar Front (APF), deep-water troughs and distance between sites. Mitochondrial DNA (mtDNA) data indicated a sharp genetic division between the Patagonian Shelf/North Scotia Ridge and the Shag Rocks/South Georgia samples, whereas microsatellite data showed much less distinct structuring and an intermediate position of the North Scotia Ridge samples. We suggest these data indicate that the APF, as a barrier to larval dispersal, is the major inhibitor of genetic exchange between toothfish populations, with deep-water troughs and distance between sites contributing to genetic differentiation by inhibiting migration of relatively sedentary adults. We also suggest that differences between mtDNA and nuclear DNA population patterns may reflect either genome population size effects or (putative) male-biased dispersal.