Increased IGF-II protein affects p57kip2 expression in vivo and in vitro: Implications for Beckwith–Wiedemann syndrome

In both human and mouse, the Igf2 gene, localized on chromosomes 11 and 7, respectively, is expressed from the paternally inherited chromosome in the majority of tissues. Insulin-like growth factor-II (IGF-II) plays an important role in embryonic growth, and aberrant IGF2 expression has been documented in several human pathologies, such as Beckwith–Wiedemann syndrome (BWS), and a wide variety of tumors. Human and mouse genetic data strongly implicate another gene, CDKN1C (p57kip2), located in the same imprinted gene cluster on human chromosome II, in BWS. p57KIP2 is a cyclin-dependent kinase inhibitor and is required for normal mouse embryonic development. Mutations in CDKN1C (p57kip2) have been identified in a small proportion of patients with BWS, and removal of the gene from mice by targeted mutagenesis produces a phenotype with elements in common with this overgrowth syndrome. Patients with BWS with biallelic expression of IGF2 or with a CDKN1C (p57kip2) mutation, as well as overlapping phenotypes observed in two types of mutant mice, the p57kip2 knockout and IGF-II-overexpressing mice, strongly suggest that the genes may act in a common pathway of growth control in situations where Igf2 expression is abnormal. Herein, we show that p57kip2 expression is reduced on IGF-II treatment of primary embryo fibroblasts in a dose-dependent manner. In addition, p57kip2 expression is down-regulated in mice with high serum levels of IGF-II. These data suggest that the effects of increased IGF-II in BWS may, in part, be mediated through a decrease in p57kip2 gene expression.

Identification of the yeast cytidine deaminase CDD1 as an orphan C���U RNA editase

Yeast co-expressing rat APOBEC-1 and a fragment of human apolipoprotein B (apoB) mRNA assembled functional editosomes and deaminated C6666 to U in a mooring sequence-dependent fashion. The occurrence of APOBEC-1-complementing proteins suggested a naturally occurring mRNA editing mechanism in yeast. Previously, a hidden Markov model identified seven yeast genes encoding proteins possessing putative zinc-dependent deaminase motifs. Here, only CDD1, a cytidine deaminase, is shown to have the capacity to carry out C���U editing on a reporter mRNA. This is only the second report of a cytidine deaminase that can use mRNA as a substrate. CDD1-dependent editing was growth phase regulated and demonstrated mooring sequence-dependent editing activity. Candidate yeast mRNA substrates were identified based on their homology with the mooring sequence-containing tripartite motif at the editing site of apoB mRNA and their ability to be edited by ectopically expressed APOBEC-1. Naturally occurring yeast mRNAs edited to a significant extent by CDD1 were, however, not detected. We propose that CDD1 be designated an orphan C���U editase until its native RNA substrate, if any, can be identified and that it be added to the CDAR (cytidine deaminase acting on RNA) family of editing enzymes.

Microvascular and tissue oxygen gradients in the rat mesentery

One of the most important functions of the blood circulation is O2 delivery to the tissue. This process occurs primarily in microvessels that also regulate blood flow and are the site of many metabolic processes that require O2. We measured the intraluminal and perivascular pO2 in rat mesenteric arterioles in vivo by using noninvasive phosphorescence quenching microscopy. From these measurements, we calculated the rate at which O2 diffuses out of microvessels from the blood. The rate of O2 efflux and the O2 gradients found in the immediate vicinity of arterioles indicate the presence of a large O2 sink at the interface between blood and tissue, a region that includes smooth muscle and endothelium. Mass balance analyses show that the loss of O2 from the arterioles in this vascular bed primarily is caused by O2 consumption in the microvascular wall. The high metabolic rate of the vessel wall relative to parenchymal tissue in the rat mesentery suggests that in addition to serving as a conduit for the delivery of O2 the microvasculature has other functions that require a significant amount of O2.

Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects

Yeast phosphatidylinositol transfer protein (Sec14p) function is essential for production of Golgi-derived secretory vesicles, and this requirement is bypassed by mutations in at least seven genes. Analyses of such ‘bypass Sec14p’ mutants suggest that Sec14p acts to maintain an essential Golgi membrane diacylglycerol (DAG) pool that somehow acts to promote Golgi secretory function. SPO14 encodes the sole yeast phosphatidylinositol-4,5-bisphosphate-activated phospholipase D (PLD). PLD function, while essential for meiosis, is dispensable for vegetative growth. Herein, we report specific physiological circumstances under which an unanticipated requirement for PLD activity in yeast vegetative Golgi secretory function is revealed. This PLD involvement is essential in ‘bypass Sec14p’ mutants where normally Sec14p-dependent Golgi secretory reactions are occurring in a Sec14p-independent manner. PLD catalytic activity is necessary but not sufficient for ‘bypass Sec14p’, and yeast operating under ‘bypass Sec14p’ conditions are ethanol-sensitive. These data suggest that PLD supports ‘bypass Sec14p’ by generating a phosphatidic acid pool that is somehow utilized in supporting yeast Golgi secretory function.

Anteroposterior neural tissue specification by activin-induced mesoderm

The transforming growth factor β superfamily member, activin, is able to induce mesodermal tissues in animal cap explants from Xenopus laevis blastula stage embryos. Activin can act like a morphogen of the dorsoventral axis in that lower doses induce more ventral, and higher doses more dorsal, tissue types. Activin has also previously been reported to induce neural tissues in animal caps. From cell mixing experiments it was inferred that this might be an indirect effect of induced mesoderm signaling to uninduced ectoderm. Here we demonstrate directly that neural tissues do indeed arise by the action of induced mesoderm on uninduced ectoderm. Dorsal mesoderm is itself subdivided into posterior and anterior domains in vivo, but this had not been demonstrated for induced mesoderm. We therefore tested whether different concentrations of activin recreate these different anteroposterior properties as well. We show that the anteroposterior positional value of induced mesoderm, including its neuroinductive properties, depends on the dose of activin applied to the mesoderm, with lower doses inducing more posterior and higher doses giving more anterior markers. We discuss the implications of these results for patterning signals and the relationship between anteroposterior and dorsoventral axes.

Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin-(HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1–56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.

Horizontal cells reveal cone type-specific adaptation in primate retina

The human cone visual system maintains contrast sensitivity over a wide range of ambient illumination, a property known as light adaptation. The first stage in light adaptation is believed to take place at the first neural step in vision, within the long, middle, and short wavelength sensitive cone photoreceptors. To determine the properties of adaptation in primate outer retina, we measured cone signals in second-order interneurons, the horizontal cells, of the macaque monkey. Horizontal cells provide a unique site for studying early adaptational mechanisms; they are but one synapse away from the photoreceptors, and each horizontal cell receives excitatory inputs from many cones. Light adaptation occurred over the entire range of light levels evaluated, a luminance range of 15–1,850 trolands. Adaptation was demonstrated to be independent in each cone type and to be spatially restricted. Thus, in primates, a major source of sensitivity regulation occurs before summation of cone signals in the horizontal cell.

Polymerizing Microtubules Activate Site-directed F-Actin Assembly in Nerve Growth Cones

We identify an actin-based protrusive structure in growth cones termed “intrapodium.” Unlike filopodia, intrapodia are initiated exclusively within lamellipodia and elongate in a continuous (nonsaltatory) manner parallel to the plane of the dorsal plasma membrane causing a ridge-like protrusion. Intrapodia resemble the actin-rich structures induced by intracellular pathogens (e.g., Listeria) or by extracellular beads. Cytochalasin B inhibits intrapodial elongation and removal of cytochalasin B produced a burst of intrapodial activity. Electron microscopic studies revealed that lamellipodial intrapodia contain both short and long actin filaments oriented with their barbed ends toward the membrane surface or advancing end. Our data suggest an interaction between microtubule endings and intrapodia formation. Disruption of microtubules by acute nocodazole treatment decreased intrapodia frequency, and washout of nocodazole or addition of the microtubule-stabilizing drug Taxol caused a burst of intrapodia formation. Furthermore, individual microtubule ends were found near intrapodia initiation sites. Thus, microtubule ends or associated structures may regulate these actin-dependent structures. We propose that intrapodia are the consequence of an early step in a cascade of events that leads to the development of F-actin-associated plasma membrane specializations.

Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication

New antibiotics to combat the emerging pandemic of drug-resistant strains of Mycobacterium tuberculosis are urgently needed. We have investigated the effects on M. tuberculosis of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of glutamine synthetase, an enzyme whose export is associated with pathogenicity and with the formation of a poly-l-glutamate/glutamine cell wall structure. Treatment of virulent M. tuberculosis with 10 μM antisense PS-ODNs reduced glutamine synthetase activity and expression by 25–50% depending on whether one, two, or three different PS-ODNs were used and the PS-ODNs' specific target sites on the mRNA. Treatment with PS-ODNs of a recombinant strain of Mycobacterium smegmatis expressing M. tuberculosis glutamine synthetase selectively inhibited the recombinant enzyme but not the endogenous enzyme for which the mRNA transcript was mismatched by 2–4 nt. Treatment of M. tuberculosis with the antisense PS-ODNs also reduced the amount of poly-l-glutamate/glutamine in the cell wall by 24%. Finally, treatment with antisense PS-ODNs reduced M. tuberculosis growth by 0.7 logs (1 PS-ODN) to 1.25 logs (3 PS-ODNs) but had no effect on the growth of M. smegmatis, which does not export glutamine synthetase nor possess the poly-l-glutamate/glutamine (P-l-glx) cell wall structure. The experiments indicate that the antisense PS-ODNs enter the cytoplasm of M. tuberculosis and bind to their cognate targets. Although more potent ODN technology is needed, this study demonstrates the feasibility of using antisense ODNs in the antibiotic armamentarium against M. tuberculosis.

Evolutionary relationships among diverse bacteriophages and prophages: All the world’s a phage

We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage φC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, φflu, in the Haemophilus influenzae genome, and two small prophage-like elements, φRv1 and φRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.

Solvent dependence of dynamic transitions in protein solutions

A transition as a function of increasing temperature from harmonic to anharmonic dynamics has been observed in globular proteins by using spectroscopic, scattering, and computer simulation techniques. We present here results of a dynamic neutron scattering analysis of the solvent dependence of the picosecond-time scale dynamic transition behavior of solutions of a simple single-subunit enzyme, xylanase. The protein is examined in powder form, in D2O, and in four two-component perdeuterated single-phase cryosolvents in which it is active and stable. The scattering profiles of the mixed solvent systems in the absence of protein are also determined. The general features of the dynamic transition behavior of the protein solutions follow those of the solvents. The dynamic transition in all of the mixed cryosolvent–protein systems is much more gradual than in pure D2O, consistent with a distribution of energy barriers. The differences between the dynamic behaviors of the various cryosolvent protein solutions themselves are remarkably small. The results are consistent with a picture in which the picosecond-time scale atomic dynamics respond strongly to melting of pure water solvent but are relatively invariant in cryosolvents of differing compositions and melting points.

Coupling of histamine H3 receptors to neuronal Na+/H+ exchange: A novel protective mechanism in myocardial ischemia

In myocardial ischemia, adrenergic nerves release excessive amounts of norepinephrine (NE), causing dysfunction and arrhythmias. With anoxia and the concomitant ATP depletion, vesicular storage of NE is impaired, resulting in accumulation of free NE in the axoplasm of sympathetic nerves. Intraneuronal acidosis activates the Na+/H+ exchanger (NHE), leading to increased Na+ entry in the nerve terminals. These conditions favor availability of the NE transporter to the axoplasmic side of the membrane, causing massive carrier-mediated efflux of free NE. Neuronal NHE activation is pivotal in this process; NHE inhibitors attenuate carrier-mediated NE release. We previously reported that activation of histamine H3 receptors (H3R) on cardiac sympathetic nerves also reduces carrier-mediated NE release and alleviates arrhythmias. Thus, H3R activation may be negatively coupled to NHE. We tested this hypothesis in individual human SKNMC neuroblastoma cells stably transfected with H3R cDNA, loaded with the intracellular pH (pHi) indicator BCECF. These cells possess amiloride-sensitive NHE. NHE activity was measured as the rate of Na+-dependent pHi recovery in response to an acute acid pulse (NH4Cl). We found that the selective H3R-agonist imetit markedly diminished NHE activity, and so did the amiloride derivative EIPA. The selective H3R antagonist thioperamide abolished the imetit-induced NHE attenuation. Thus, our results provide a link between H3R and NHE, which may limit the excessive release of NE during protracted myocardial ischemia. Our previous and present findings uncover a novel mechanism of cardioprotection: NHE inhibition in cardiac adrenergic neurons as a means to prevent ischemic arrhythmias associated with carrier-mediated NE release.