microRNA-34a is tumor suppressive in brain tumors and glioma stem cells

We recently found that microRNA-34a (miR-34a) is downregulated in human glioma tumors as compared to normal brain, and that miR-34a levels in mutant-p53 gliomas were lower than in wildtype-p53 tumors. We showed that miR-34a expression in glioma and medulloblastoma cells inhibits cell proliferation, G1/S cell cycle progression, cell survival, cell migration and cell invasion, but that miR-34a expression in human astrocytes does not affect cell survival and cell cycle. We uncovered the oncogenes c-Met, Notch-1 and Notch-2 as direct targets of miR-34a that are inhibited by miR-34a transfection. We found that c-Met levels in human glioma specimens inversely correlate with miR-34a levels. We showed that c-Met and Notch partially mediate the inhibitory effects of miR-34a on cell proliferation and cell death. We also found that mir-34a expression inhibits in vivo glioma xenograft growth. We concluded that miR-34a is a potential tumor suppressor in brain tumors that acts by targeting multiple oncogenes. In this extra view, we briefly review and discuss the implications of these findings and present new data on the effects of miR-34a in glioma stem cells. The new data show that miR-34a expression inhibits various malignancy endpoints in glioma stem cells. Importantly, they also show for the first time that miR-34a expression induces glioma stem cell differentiation. Altogether, the data suggest that miR-34a is a tumor suppressor and a potential potent therapeutic agent that acts by targeting multiple oncogenic pathways in brain tumors and by inducing the differentiation of cancer stem cells.     

Relative rates of synonymous substitutions in the mitochondrial, chloroplast and nuclear genomes of seed plants

Previous studies have estimated that, in angiosperms, the synonymous substitution rate of chloroplast genes is three times higher than that of mitochondrial genes and that of nuclear genes is twelve times higher than that of mitochondrial genes. Here we used 12 genes in 27 seed plant species to investigate whether these relative rates of substitutions are common to diverse seed plant groups. We find that the overall relative rate of synonymous substitutions of mitochondrial, chloroplast and nuclear genes of all seed plants is 1:3:10, that these ratios are 1:2:4 in gymnosperms but 1:3:16 in angiosperms and that they go up to 1:3:20 in basal angiosperms. Our results show that the mitochondrial, chloroplast and nuclear genomes of seed plant groups have different synonymous substitutions rates, that these rates are different in different seed plant groups and that gymnosperms have smaller ratios than angiosperms.     

Tau phosphorylation: physiological and pathological consequences

The microtubule-associated protein tau, abundant in neurons, has gained notoriety due to the fact that it is deposited in cells as fibrillar lesions in numerous neurodegenerative diseases, and most notably Alzheimer's disease. Regulation of microtubule dynamics is the most well-recognized function of tau, but it is becoming increasingly evident that tau plays additional roles in the cell. The functions of tau are regulated by site-specific phosphorylation events, which if dysregulated, as they are in the disease state, result in tau dysfunction and mislocalization, which is potentially followed by tau polymerization, neuronal dysfunction and death. Given the increasing evidence that a disruption in the normal phosphorylation state of tau plays a key role in the pathogenic events that occur in Alzheimer's disease and other neurodegenerative conditions, it is of crucial importance that the protein kinases and phosphatases that regulate tau phosphorylation in vivo as well as the signaling cascades that regulate them be identified. This review focuses on recent literature pertaining to the regulation of tau phosphorylation and function in cell culture and animal model systems, and the role that a dysregulation of tau phosphorylation may play in the neuronal dysfunction and death that occur in neurodegenerative diseases that have tau pathology.     

Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa

Plant root development is an essential determinant of plant growth and crop yield that could be enhanced by induced changes in the expression of root-specific regulatory factors. We reported previously that Alfin1 binds DNA in a sequence-specific manner and that Alfin1 overexpression in transgenic alfalfa (Medicago sativa L.) enhances expression of the salt-inducible MsPRP2 gene in roots, suggesting that Alfin1 functions to regulate gene expression in roots. Here we show that Alfin1 is an essential gene for root growth and that its overexpression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions. Alfin1-binding sites occur in promoters of genes expressed in roots of a wide variety of plant species and we propose that it is a general root growth regulator. Even though Alfin1 overexpression was under the control of the CaMV 35S promoter, plant shoot growth was not adversely affected. We show further that introduction of the Alfin1 transgene in plants confers a dominant characteristic that significantly increases plant growth and salt tolerance.     

Absence of Spatial Tuning in the Orbitofrontal Cortex

There is limited data in the literature to explicitly support the notion that neurons in OFC are truly action-independent in their coding. We set out to specifically test the hypothesis that OFC value-related neurons in area 13 m of the monkey do not carry information about the action required to obtain that reward – that activity in this area represents reward values in an abstract and action-independent manner. To accomplish that goal we had two monkeys select and execute saccadic eye movements to 81 locations in the visual field for three different kinds of juice rewards. Our detailed analysis of the response fields indicates that these neurons are insensitive to the amplitude or direction of the saccade required to obtain these rewards. Our data thus validate earlier proposals that neurons of 13 m in the OFC encode subjective value independent of the saccadic action required to obtain that reward.     

The neural and cognitive correlates of aimed throwing in chimpanzees: a magnetic resonance image and behavioural study on a unique form of social tool use

It has been hypothesized that neurological adaptations associated with evolutionary selection for throwing may have served as a precursor for the emergence of language and speech in early hominins. Although there are reports of individual differences in aimed throwing in wild and captive apes, to date there has not been a single study that has examined the potential neuroanatomical correlates of this very unique tool-use behaviour in non-human primates. In this study, we examined whether differences in the ratio of white (WM) to grey matter (GM) were evident in the homologue to Broca's area as well as the motor-hand area of the precentral gyrus (termed the KNOB) in chimpanzees that reliably throw compared with those that do not. We found that the proportion of WM in Broca's homologue and the KNOB was significantly higher in subjects that reliably throw compared with those that do not. We further found that asymmetries in WM within both brain regions were larger in the hemisphere contralateral to the chimpanzee's preferred throwing hand. We also found that chimpanzees that reliably throw show significantly better communication abilities than chimpanzees that do not. These results suggest that chimpanzees that have learned to throw have developed greater cortical connectivity between primary motor cortex and the Broca's area homologue. It is suggested that during hominin evolution, after the split between the lines leading to chimpanzees and humans, there was intense selection on increased motor skills associated with throwing and that this potentially formed the foundation for left hemisphere specialization associated with language and speech found in modern humans.     

Dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet

Recent studies have shown that dietary creatine supplementation can prevent lipid accumulation in the liver. Creatine is a small molecule that plays a large role in energy metabolism, but since the enzyme creatine kinase is not present in the liver, the classical role in energy metabolism does not hold in this tissue. Fat accumulation in the liver can lead to the development of nonalcoholic fatty liver disease (NAFLD), a progressive disease that is prevalent in humans. We have previously reported that creatine can directly influence lipid metabolism in cell culture to promote lipid secretion and oxidation. Our goal in the current study was to determine whether similar mechanisms that occur in cell culture were present in vivo. We also sought to determine whether dietary creatine supplementation could be effective in reversing steatosis. Sprague–Dawley rats were fed a high-fat diet or a high-fat diet supplemented with creatine for 5 weeks. We found that rats supplemented with creatine had significantly improved rates of lipoprotein secretion and alterations in mitochondrial function that were consistent with greater oxidative capacity. We also find that introducing creatine into a high-fat diet halted hepatic lipid accumulation in rats with fatty liver. Our results support our previous report that liver cells in culture with creatine secrete and oxidize more oleic acid, demonstrating that dietary creatine can effectively change hepatic lipid metabolism by increasing lipoprotein secretion and oxidation in vivo. Our data suggest that creatine might be an effective therapy for NAFLD.     

Extracellular superoxide dismutase inhibits inflammation by preventing oxidative fragmentation of hyaluronan

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.     

The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis.

The mechanisms that regulate the growth of the brain remain unclear. We show that Sonic hedgehog (Shh) is expressed in a layer-specific manner in the perinatal mouse neocortex and tectum, whereas the Gli genes, which are targets and mediators of SHH signaling, are expressed in proliferative zones. In vitro and in vivo assays show that SHH is a mitogen for neocortical and tectal precursors and that it modulates cell proliferation in the dorsal brain. Together with its role in the cerebellum, our findings indicate that SHH signaling unexpectedly controls the development of the three major dorsal brain structures. We also show that a variety of primary human brain tumors and tumor lines consistently express the GLI genes and that cyclopamine, a SHH signaling inhibitor, inhibits the proliferation of tumor cells. Using the in vivo tadpole assay system, we further show that misexpression of GLI1 induces CNS hyperproliferation that depends on the activation of endogenous Gli1 function. SHH-GLI signaling thus modulates normal dorsal brain growth by controlling precursor proliferation, an evolutionarily important and plastic process that is deregulated in brain tumors.     

Functional specialization of ribosomes?

Ribosomes are highly conserved macromolecular machines that are responsible for protein synthesis in all living organisms. Work published in the past year has shown that changes to the ribosome core can affect the mechanism of translation initiation that is favored in the cell, which potentially leads to specific changes in the relative efficiencies with which different proteins are made. Here, I examine recent data from expression and proteomic studies that suggest that cells make slightly different ribosomes under different growth conditions, and discuss genetic evidence that such differences are functional. In particular, I argue that eukaryotic cells probably produce ribosomes that lack one or more core ribosomal proteins (RPs) under some conditions, and that core RPs contribute differentially to translation of distinct subpopulations of mRNAs.     

Analysis of cellular factors that mediate nuclear export of RNAs bearing the Mason-Pfizer monkey virus constitutive transport element

There is now convincing evidence that the human Tap protein plays a critical role in mediating the nuclear export of mRNAs that contain the Mason-Pfizer monkey virus constitutive transport element (CTE) and significant evidence that Tap also participates in global poly(A)(+) RNA export. Previously, we had mapped carboxy-terminal sequences in Tap that serve as an essential nucleocytoplasmic shuttling domain, while others had defined an overlapping Tap sequence that can bind to the FG repeat domains of certain nucleoporins. Here, we demonstrate that these two biological activities are functionally correlated. Specifically, mutations in Tap that block nucleoporin binding also block both nucleocytoplasmic shuttling and the Tap-dependent nuclear export of CTE-containing RNAs. In contrast, mutations that do not inhibit nucleoporin binding also fail to affect Tap shuttling. Together, these data indicate that Tap belongs to a novel class of RNA export factors that can target bound RNA molecules directly to the nuclear pore without the assistance of an importin beta-like cofactor. In addition to nucleoporins, Tap has also been proposed to interact with a cellular cofactor termed p15. Although we were able to confirm that Tap can indeed bind p15 specifically both in vivo and in vitro, a mutation in Tap that blocked p15 binding only modestly inhibited CTE-dependent nuclear RNA export. However, p15 did significantly enhance the affinity of Tap for the CTE in vitro and readily formed a ternary complex with Tap on the CTE. This result suggests that p15 may play a significant role in the recruitment of the Tap nuclear export factor to target RNA molecules in vivo.     

Protein kinase A phosphorylation of the cardiac calcium release channel (ryanodine receptor) in normal and failing hearts. Role of phosphatases and response to isoproterenol

The cardiac ryanodine receptor/calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) comprises a macromolecular complex that includes a kinase and two phosphatases that are bound to the channel via targeting proteins. We previously found that the RyR2 is protein kinase A (PKA)-hyperphosphorylated in end-stage human heart failure. Because heart failure is a progressive disease that often evolves from hypertrophy, we analyzed the RyR2 macromolecular complex in several animal models of cardiomyopathy that lead to heart failure, including hypertrophy, and at different stages of disease progression. We now show that RyR2 is PKA-hyperphosphorylated in diverse models of heart failure and that the degree of RyR2 PKA phosphorylation correlates with the degree of cardiac dysfunction. Interestingly, we show that RyR2 PKA hyperphosphorylation can be lost during perfusion of isolated hearts due to the activity of the endogenous phosphatases in the RyR2 macromolecular complex. Moreover, infusion of isoproterenol resulted in PKA phosphorylation of RyR2 in rat, indicating that systemic catecholamines can activate phosphorylation of RyR2 in vivo. These studies extend our previous analyses of the RyR2 macromolecular complex, show that both the kinase and phosphatase activities in the macromolecular complex are regulated physiologically in vivo, and suggest that RyR2 PKA hyperphosphorylation is likely a general feature of heart failure.     

Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis

It is generally thought that mucosal bacterial pathogens of the genera Haemophilus, Neisseria, and Moraxella elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains. Haemophilus influenzae elaborates short-chain LPS that has a role in the pathogenesis of H. influenzae infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the H. influenzae genome, the hmg locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.     

Regulation of aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii compared to Saccharomyces cerevisiae

To elucidate the growth inhibitory effect of threonine, the regulation of the aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii, an important yeast for the flavor development in soy sauce, was investigated. It was shown that threonine inhibited the growth of Z. rouxii by blocking the methionine synthesis. It seemed that threonine blocked this synthesis by inhibiting the conversion of aspartate. In addition, it was shown that the growth of Z. rouxii, unlike that of Saccharomyces cerevisiae, was not inhibited by the herbicide sulfometuron methyl (SMM). From enzyme assays, it was concluded that the acetohydroxy acid synthase in Z. rouxii, unlike that in S. cerevisiae, was not sensitive to SMM. Furthermore, the enzyme assays demonstrated that the activity of threonine deaminase in Z. rouxii, like in S. cerevisiae, was strongly inhibited by isoleucine and stimulated by valine. From this work, it is clear that the aspartate-derived amino-acid metabolism in Z. rouxii only partly resembles that in S. cerevisiae.     

Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins

Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-beta-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute gamma-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.     

Synonymous Substitution Rates in Enterobacteria

It has been shown previously that the synonymous substitution rate between Escherichia coli and Salmonella typhimurium is lower in highly than in weakly expressed genes, and it has been suggested that this is due to stronger selection for translational efficiency in highly expressed genes as reflected in their greater codon usage bias. This hypothesis is tested here by comparing the substitution rate in codon families with different patterns of synonymous codon use. It is shown that the decline in the substitution rate across expression levels is as great for codon families that do not appear to be subject to selection for translational efficiency as for those that are. This implies that selection on translational efficiency is not responsible for the decline in the substitution rate across genes. It is argued that the most likely explanation for this decline is a decrease in the mutation rate. It is also shown that a simple evolutionary model in which synonymous codon use is determined by a balance between mutation, selection for an optimal codon, and genetic drift predicts that selection should have little effect on the substitution rate in the present case.     

Splice junctions are constrained by protein disorder

We have discovered that positions of splice junctions in genes are constrained by the tolerance for disorder-promoting amino acids in the translated protein region. It is known that efficient splicing requires nucleotide bias at the splice junction; the preferred usage produces a distribution of amino acids that is disorder-promoting. We observe that efficiency of splicing, as seen in the amino-acid distribution, is not compromised to accommodate globular structure. Thus we infer that it is the positions of splice junctions in the gene that must be under constraint by the local protein environment. Examining exonic splicing enhancers found near the splice junction in the gene, reveals that these (short DNA motifs) are more prevalent in exons that encode disordered protein regions than exons encoding structured regions. Thus we also conclude that local protein features constrain efficient splicing more in structure than in disorder.