Samuel E. Lux

Positional cloning of zebrafish ferroportin1 identifies a conservedvertebrate iron exporter

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMT1 (refs 1,2,3). A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

A specific apoprotein activator for lipoprotein lipase

Abstract These studies were designed to determine which of the apoproteins of high density lipoprotein (HDL) function as the cofactor for lipoprotein lipase (LPL). ApoLP-gln and apoLP-thr, the major HDL apoproteins, as well as apoLP-val, a minor apoprotein constituent, are inactive as cofactors even in the presence of phospholipid. ApoLP-ala, another minor constituent, is inactive alone but in the presence of phospholipid stimulates lipase activity twofold. Only apoLP-glu is able to stimulate LPL activity in the absence of phospholipid and, in the presence of phospholipid, increases activity twelvefold over baseline levels. It is possible that apoLP-glu and perhaps apoLP-ala are obligatory “co-factors” for the hydrolytic step required for normal clearing of triglyceride from the plasma.

Dependence of Nodal Sodium Channel Clustering on Paranodal Axoglial Contact in the Developing CNS

Na+ channel clustering at nodes of Ranvier in the developing rat optic nerve was analyzed to determine mechanisms of localization, including the possible requirement for glial contactin vivo. Immunofluorescence labeling for myelin-associated glycoprotein and for the protein Caspr, a component of axoglial junctions, indicated that oligodendrocytes were present, and paranodal structures formed, as early as postnatal day 7 (P7). However, the first Na+ channel clusters were not seen until P9. Most of these were broad, and all were excluded from paranodal regions of axoglial contact. The number of detected Na+ channel clusters increased rapidly from P12 to P22. During this same period, conduction velocity increased sharply, and Na+ channel clusters became much more focal. To test further whether oligodendrocyte contact directly influences Na+ channel distributions, nodes of Ranvier in the hypomyelinating mouse Shiverer were examined. This mutant has oligodendrocyte-ensheathed axons but lacks compact myelin and normal axoglial junctions. During development Na+ channel clusters in Shiverer mice were reduced in numbers and were in aberrant locations. The subcellular location of Caspr was disrupted, and nerve conduction properties remained immature. These results indicate that in vivo, Na+ channel clustering at nodes depends not only on the presence of oligodendrocytes but also on specific axoglial contact at paranodal junctions. In rats, ankyrin-3/G, a cytoskeletal protein implicated in Na+ channel clustering, was detected before Na+ channel immunoreactivity but extended into paranodes in non-nodal distributions. In Shiverer, ankyrin-3/G labeling was abnormal, suggesting that its localization also depends on axoglial contact.

ANION EXCHANGER 1 (BAND 3) IS REQUIRED TO PREVENT ERYTHROCYTE MEMBRANE SURFACE LOSS BUT NOT TO FORM THE MEMBRANE SKELETON

The red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1-lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis.

Notch1 inhibits neurite outgrowth in postmitotic primary neurons

Notch plays an important role in cell fate decisions in uncommitted proliferative cells, including neurogenesis, but is believed to not have a role in postmitotic cells. We have shown previously that Notch1 is highly expressed in embryonal mouse and human brain, but surprisingly it continues to be expressed at low levels in the adult brain. The function of Notch1 in postmitotic neurons in mammals is unknown. To better understand the potential role of Notch1 in mature central nervous system neurons we studied the effect of Notch1 transfection on neurite outgrowth in primary neocortex hippocampal neurons. Transfection at two days in vitro with full length Notch1 inhibited neurite outgrowth. Transfection at five to six days in vitro, after neurite outgrowth was established, led to apparent regression of neurites. These effects were enhanced when truncated constitutively active forms of Notch1 were introduced. Co-transfection with Numb, a physiological inhibitor of Notch, blocked Notchs effect on neurite outgrowth. We also examined whether Notch1 could activate C-promoter binding factor (CBF1) transcription factor using C-promoter binding factor-luciferase constructs, and demonstrated that this signal transduction pathway is present and can be activated in postmitotic neurons. Our results show that in postmitotic neurons Notch1 influences neurite morphology, and can activate its native signal transduction pathway. These data strongly suggest that Notch1 may play a physiologically important role in the central nervous system beyond neurogenesis.

Dietary and Drug Treatment of Primary Hyperlipoproteinemia

Abstract The first step in the management of primary hyperlipidemia is its translation into hyperlipoproteinemia, which can be done by measuring the plasma cholesterol and triglyceride concentratio...

Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency.

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R–binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.

Electron microscopic study on reassembly of plasma high density apoprotein with various lipids.

Products resulting from the sonification of mixtures of plasma high density lipoprotein apoprotein and specific lipids were studied by electron microscopy using negative staining. Sonicates of apoprotein plus lecithin produced disc-shaped structures which stacked in aggregates with a 50–55-A repeat; the discs were 100–200 A in diameter. Incorporation of unesterified cholesterol into the mixture produced structures morphologically similar to those observed in sonicates of apoprotein plus lecithin. Disc-shaped particles from sonified mixtures of apoprotein, lecithin and unesterified cholesterol were ultracentrifugally isolated in the d 1.063–1.21 g/ml fraction and were incubated with a plasma d > 1.21 g/ml fraction containing lecithin: cholesterol acyltransferase activity. Electron microscopy following the incubation procedure showed a transformation of the disc-like structures into approximately spherical particles (50–100 A diameter). Similar spherical particles were also obtained after sonification of apoprotein-lecithin-unesterified cholesterol-cholesteryl ester mixtures. Results indicate a requirement for the presence of cholesteryl esters to maintain normal morphology of plasma high density lipoproteins.

Chronic Neutropenia and Abnormal Cellular Immunity in Cartilage-Hair Hypoplasia

Abstract Two children with cartilage-hair hypoplasia suffered from recurrent respiratory-tract infections and contracted unusually severe varicella. Hematologic studies in one child disclosed chron...

A Genetic Defect in the Binding of Protein 4.1 to Spectrin in a Kindred with Hereditary Spherocytosis

Indirect evidence suggests that the genetic defect in hereditary spherocytosis lies in the erythrocyte membrane skeleton, a submembranous meshwork of proteins (principally spectrin, actin, and protein 4.1) responsible for membrane shape and structural stability. To test this premise we systematically assayed the interactions of spectrin, the major skeletal protein, in six kindreds with autosomal dominant hereditary spherocytosis. In one these kindreds, enhancement of spectrin-actin binding by protein 4.1 was reduced, owing to a 39 +/- 4 per cent decrease (mean +/- S.D) in the binding of normal protein 4.1 by spectrin, in all of four members with the disorder. The defective spectrin was separated into two populations by affinity chromatography on immobilized normal protein 4.1. One population (41 +/- 2 per cent) lacked the ability to bind 4.1, but the other functioned normally. Presumable, the nonfunctional spectrin was the product of the autosomal dominant gene responsible for the hereditary spherocytosis in this kindred.