Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice.

In mammalian cells, regulation of the expression of proteins involved in iron metabolism is achieved through interactions of iron-sensing proteins known as iron regulatory proteins (IRPs), with transcripts that contain RNA stem-loop structures referred to as iron responsive elements (IREs). Two distinct but highly homologous proteins, IRP1 and IRP2, bind IREs with high affinity when cells are depleted of iron, inhibiting translation of some transcripts, such as ferritin, or turnover of others, such as the transferrin receptor (TFRC). IRPs sense cytosolic iron levels and modify expression of proteins involved in iron uptake, export and sequestration according to the needs of individual cells. Here we generate mice with a targeted disruption of the gene encoding Irp2 (Ireb2). These mutant mice misregulate iron metabolism in the intestinal mucosa and the central nervous system. In adulthood, Ireb2(-/-) mice develop a movement disorder characterized by ataxia, bradykinesia and tremor. Significant accumulations of iron in white matter tracts and nuclei throughout the brain precede the onset of neurodegeneration and movement disorder symptoms by many months. Ferric iron accumulates in the cytosol of neurons and oligodendrocytes in distinctive regions of the brain. Abnormal accumulations of ferritin colocalize with iron accumulations in populations of neurons that degenerate, and iron-laden oligodendrocytes accumulate ubiquitin-positive inclusions. Thus, misregulation of iron metabolism leads to neurodegenerative disease in Ireb2(-/-) mice and may contribute to the pathogenesis of comparable human neurodegenerative diseases.

Thyrotropin-releasing hormone precursor: characterization in rat brain.

To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.

Primary structure and functional expression of a mammalian skeletal muscle sodium channel.

We describe the isolation and characterization of a cDNA encoding the alpha subunit of a new voltage-sensitive sodium channel, microI, from rat skeletal muscle. The 1840 amino acid microI peptide is homologous to alpha subunits from rat brain, but, like the protein from eel electroplax, lacks an extended (approximately 200) amino acid segment between homologous domains I and II. Northern blot analysis indicates that the 8.5 kb microI transcript is preferentially expressed in skeletal muscle. Sodium channels expressed in Xenopus oocytes from synthetic RNA encoding microI are blocked by tetrodotoxin and mu-conotoxin at concentrations near 5 nM. The expressed sodium channels have gating kinetics similar to the native channels in rat muscle fibers, except that inactivation occurs more slowly.

Expression of diverse Na+ channel messenger RNAs in rat myocardium. Evidence for a cardiac-specific Na+ channel.

This study examined the diversity of Na+ channel gene expression in intact cardiac tissue and purified myocardial cells. The screening of neonatal rat myocardial cell cDNA libraries with a conserved rat brain Na+ channel cDNA probe, resulted in the isolation and characterization of a putative rat cardiac Na+ channel cDNA probe (pCSC-1). The deduced amino acid sequence of pCSC-1 displayed a striking degree of homology with the eel, rat brain-1, and rat brain-2 Na+ channel, thereby identifying pCSC-1 as a related member of the family of Na+ channel genes. Northern blot analysis revealed the expression of a 7-kb CSC-1 transcript in rat cardiac tissue and purified myocardial cells, but little or no detectable expression of CSC-1 in rat brain, skeletal muscle, denervated skeletal muscle, or liver. Using RNase protection and Northern blot hybridization with specific rat brain Na+ channel gene probes, expression of the rat brain-1 Na+ channel was observed in rat myocardium, but no detectable expression of the rat brain-2 gene was found. This study provides evidence for the expression of diverse Na+ channel mRNAs in rat myocardium and presents the initial characterization of a new, related member of the family of Na+ channel genes, which appears to be expressed in a cardiac-specific manner.

An attempt to objectify the therapeutic process: a working model.

On the basis of their observations in daily psychoanalytic work the authors developed five objective criteria for "bad" analytic hours. These criteria involve affect, intellectualization, isolation, lack of feedback, and dissatisfaction. The authors developed a formulation to help them preconsciously recognize the presence of these factors during therapeutic work and found that it was helpful in turning a potentially bad hour into a productive one.

Affective predictors of preschoolers' aggression and peer acceptance: direct and indirect effects.

Observational assessments were made of 51 preschoolers' (mean age = 53.25 months) peer aggression and emotional displays outside of (baseline) and during aggressive interactions, and their emotion knowledge and peer acceptance were also assessed. Results indicated that the connections between children's affective dispositions and their aggression and peer acceptance varied as a function of both the emotion context (baseline vs. aggression related) and the particular emotion involved (happiness vs. anger). Emotion knowledge and affective dispositions overlapped little with each other, and both made independent contributions to peer acceptance and aggression. Mediation analyses revealed, however, that the significant connections between children's emotional dispositions and knowledge and their peer acceptance were mostly mediated by aggression.

Rabbit polymorphonuclear leukocytes do not secrete endogenous pyrogens or interleukin 1 when stimulated by endotoxin, polyinosine:polycytosine, or muramyl dipeptide.

Rabbit polymorphonuclear leukocytes were purified from rabbit blood by centrifugation on colloidal silica gradients followed by sedimentation in 4% Ficoll. The purified neutrophils had normal random motility, responded to chemotactic stimuli, phagocytosed zymosan particles, made superoxide, and phagocytosed and killed bacteria. However, they did not secret endogenous pyrogens either spontaneously or in response to stimulation with endotoxin, polyinosine:polycytosine, or muramyl dipeptide. Macrophages isolated on the same gradients secreted some pyrogen spontaneously and secreted considerably more in response to the same three stimuli. This evidence reinforces the idea that macrophages are the only source of endogenous pyrogens, and that pyrogens secreted by cell populations that are rich in neutrophils are to be attributed to the monocytes or macrophages that the cell populations contain.

Regulation of muscle sodium channel transcripts during development and in response to denervation.

We have recently described the cloning and functional expression of a new sodium channel subtype, microI, isolated from a denervated rat skeletal muscle cDNA library. In studies described here, we have used RNase protection and Northern blot analyses to examine the expression of microI mRNA in different tissues and in neonatal, adult, and adult denervated muscle. We found that microI transcripts were not expressed in brain or heart, or in the myogenic cell line L6, even after differentiation to myotubes. Transcripts for microI were present at low levels in neonatal skeletal muscle and increased to maximum levels in adult tissue, paralleling the expression of tetrodotoxin (TTX)-sensitive sodium currents. Surprisingly, denervation of adult muscle was also followed by a rise in microI mRNA, at a time when TTX-insensitive currents reappear. These results show that expression of this channel subtype is regulated by tissue type, development, and innervation.

Selective induction of brain type II Na+ channels by nerve growth factor.

Cells derived from a rat pheochromocytoma (PC12 cells) can generate an action potential only upon treatment with nerve growth factor. Using electrophysiological methods, we found that the appearance of action potentials in nerve growth factor-treated PC12 cells can be explained by an increase in the density of Na+ channels. The functional properties of Na+ channels in PC12 cells are similar to those described for peripheral nerves but appear to be different from Na+ channels synthesized in Xenopus oocytes injected with brain type II Na+ -channel mRNA. To determine if PC12 cells express the brain type II Na+ -channel gene, we performed RNase-protection analyses using probes that can distinguish between the brain type I and type II Na+ -channel mRNAs. The results from these studies indicate that undifferentiated PC12 cells express the type II but not the type I Na+ -channel gene. Treatment with nerve growth factor increases expression of the type II Na+ -channel gene but has no effect on type I gene expression. Our findings suggest that Na+ -channel excitability in PC12 cells is due to the specific induction of the brain type II gene by nerve growth factor.

Modulation of sodium-channel mRNA levels in rat skeletal muscle.

Action potentials in many types of excitable cells result from changes in permeability to Na ions. Although these permeability changes in nerve and muscle are mediated by voltage-gated Na channels that are functionally similar, we found that the Na-channel gene expressed in skeletal muscle is different from the genes coding for two Na channels (type I and type II) in brain. Despite the structural differences between muscle and brain Na-channel genes, a cDNA clone derived from rat brain hybridizes to skeletal muscle Na-channel mRNA of approximately 9.5 kilobases. We used this cDNA probe to measure changes in Na-channel mRNA levels in skeletal muscle during development and following denervation. By blot hybridization analysis of electrophoretically fractionated RNA, we found that Na-channel mRNA can be detected as early as embryonic day 17 and that mRNA levels increase 2-fold between birth and postnatal day 35. Denervation of adult muscle causes a further 2- to 3-fold increase in muscle Na-channel mRNA levels, suggesting that expression of Na-channel genes in fast-twitch muscle may be regulated by the state of innervation.

Peptide YY. Structure of the precursor and expression in exocrine pancreas.

Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element.

This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)-like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex from P falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.