Nerve growth factor inhibits sympathetic neurons’ response to an injury cytokine

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII

Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE. Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E. coli, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.

Targeting of a Germ Cell-specific Type 1 Hexokinase Lacking a Porin-binding Domain to the Mitochondria as Well as to the Head and Fibrous Sheath of Murine Spermatozoa

Multiple isoforms of type 1 hexokinase (HK1) are transcribed during spermatogenesis in the mouse, including at least three that are presumably germ cell specific: HK1-sa, HK1-sb, and HK1-sc. Each of these predicted proteins contains a common, germ cell-specific sequence that replaces the porin-binding domain found in somatic HK1. Although HK1 protein is present in mature sperm and is tyrosine phosphorylated, it is not known whether the various potential isoforms are differentially translated and localized within the developing germ cells and mature sperm. Using antipeptide antisera against unique regions of HK1-sa and HK1-sb, it was demonstrated that these isoforms were not found in pachytene spermatocytes, round spermatids, condensing spermatids, or sperm, suggesting that HK1-sa and HK1-sb are not translated during spermatogenesis. Immunoreactivity was detected in protein from round spermatids, condensing spermatids, and mature sperm using an antipeptide antiserum against the common, germ cell-specific region, suggesting that HK1-sc was the only germ cell-specific isoform present in these cells. Two-dimensional SDS-PAGE suggested that all of the sperm HK1-sc was tyrosine phosphorylated, and that the somatic HK1 isoform was not present. Immunoelectron microscopy revealed that HK1-sc was associated with the mitochondria and with the fibrous sheath of the flagellum and was found in discrete clusters in the region of the membranes of the sperm head. The unusual distribution of HK1-sc in sperm suggests novel functions, such as extramitochondrial energy production, and also demonstrates that a hexokinase without a classical porin-binding domain can localize to mitochondria.

Nup93, a Vertebrate Homologue of Yeast Nic96p, Forms a Complex with a Novel 205-kDa Protein and Is Required for Correct Nuclear Pore Assembly

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex–depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates.

Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

Localization of α1,3-fucosyltransferase VI in Weibel–Palade bodies of human endothelial cells

Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked α1→3 to N-acetylglucosamine. A family of α1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription–PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with β4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel–Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel–Palade bodies suggests a function independent of Golgi-associated glycosylation.

DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans

DC-SIGN, a C-type lectin expressed on the surface of dendritic cells (DCs), efficiently binds and transmits HIVs and simian immunodeficiency viruses to susceptible cells in trans. A DC-SIGN homologue, termed DC-SIGNR, has recently been described. Herein we show that DC-SIGNR, like DC-SIGN, can bind to multiple strains of HIV-1, HIV-2, and simian immunodeficiency virus and transmit these viruses to both T cell lines and human peripheral blood mononuclear cells. Binding of virus to DC-SIGNR was dependent on carbohydrate recognition. Immunostaining with a DC-SIGNR-specific antiserum showed that DC-SIGNR was expressed on sinusoidal endothelial cells in the liver and on endothelial cells in lymph node sinuses and placental villi. The presence of this efficient virus attachment factor on multiple endothelial cell types indicates that DC-SIGNR could play a role in the vertical transmission of primate lentiviruses, in the enabling of HIV to traverse the capillary endothelium in some organs, and in the presentation of virus to CD4-positive cells in multiple locations including lymph nodes.

β-Neuregulin-1 is required for the in vivo development of functional Ca2+-activated K+ channels in parasympathetic neurons

The development of functional Ca2+-activated K+ channels (KCa) in chick ciliary ganglion (CG) neurons requires interactions with afferent preganglionic nerve terminals. Here we show that the essential preganglionic differentiation factor is an isoform of β-neuregulin-1. β-Neuregulin-1 transcripts are expressed in the midbrain preganglionic Edinger–Westphal nucleus at developmental stages that coincide with or precede the normal onset of macroscopic KCa in CG neurons. Injection of β-neuregulin-1 peptide into the brains of developing embryos evoked a robust stimulation of functional KCa channels at stages before the normal appearance of these channels in CG neurons developing in vivo. Conversely, injection of a neutralizing antiserum specific for β-neuregulin-1 inhibited the development of KCa channels in CG neurons. Low concentrations of β-neuregulin-1 evoked a robust increase in whole-cell KCa in CG neurons cocultured with iris target tissues. By contrast, culturing CG neurons with iris cells or low concentrations of β-neuregulin-1 by themselves was insufficient to stimulate KCa. These data suggest that the preganglionic factor required for the development of KCa in ciliary ganglion neurons is an isoform of β-neuregulin-1, and that this factor acts in concert with target-derived trophic molecules to regulate the differentiation of excitability.

Antifreeze Proteins in Winter Rye Leaves Form Oligomeric Complexes1

Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.

Inhibitory Regulation of Higher-Plant Myosin by Ca2+ Ions1

Myosin isolated from the pollen tubes of lily (Lilium longiflorum) is composed of a 170-kD heavy chain (E. Yokota and T. Shimmen [1994] Protoplasma 177: 153–162). Both the motile activity in vitro and the F-actin-stimulated ATPase activity of this myosin were inhibited by Ca2+ at concentrations higher than 10−6 m. In the Ca2+ range between 10−6 and 10−5 m, inhibition of the motile activity was reversible. In contrast, inhibition by more than 10−5 m Ca2+ was not reversible upon Ca2+ removal. An 18-kD polypeptide that showed the same mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that of spinach calmodulin (CaM) was present in this myosin fraction. This polypeptide showed a mobility shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a Ca2+-dependent manner. Furthermore, this polypeptide was recognized by antiserum against spinach CaM. By immunoprecipitation using antiserum against the 170-kD heavy chain, the 18-kD polypeptide was coprecipitated with the 170-kD heavy chain, provided that the Ca2+ concentration was low, indicating that this 18-kD polypeptide is bound to the 170-kD myosin heavy chain. However, the 18-kD polypeptide was dissociated from the 170-kD heavy chain at high Ca2+ concentrations, which irreversibly inhibited the motile activity of this myosin. From these results, it is suggested that the 18-kD polypeptide, which is likely to be CaM, is associated with the 170-kD heavy chain as a light chain. It is also suggested that this polypeptide is involved in the regulation of this myosin by Ca2+. This is the first biochemical basis, to our knowledge, for Ca2+ regulation of cytoplasmic streaming in higher plants.

Purification and Molecular Genetic Characterization of ZPU1, a Pullulanase-Type Starch-Debranching Enzyme from Maize1

This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.

Purification and Characterization of NADP+-Linked Isocitrate Dehydrogenase from Scots Pine1 : Evidence for Different Physiological Roles of the Enzyme in Primary Development

NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic efficiency (105 m−1 s−1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.

Characterization of a Maize Tonoplast Aquaporin Expressed in Zones of Cell Division and Elongation1

We studied aquaporins in maize (Zea mays), an important crop in which numerous studies on plant water relations have been carried out. A maize cDNA, ZmTIP1, was isolated by reverse transcription-coupled PCR using conserved motifs from plant aquaporins. The derived amino acid sequence of ZmTIP1 shows 76% sequence identity with the tonoplast aquaporin γ-TIP (tonoplast intrinsic protein) from Arabidopsis. Expression of ZmTIP1 in Xenopus laevis oocytes showed that it increased the osmotic water permeability of oocytes 5-fold; this water transport was inhibited by mercuric chloride. A cross-reacting antiserum made against bean α-TIP was used for immunocytochemical localization of ZmTIP1. These results indicate that this and/or other aquaporins is abundantly present in the small vacuoles of meristematic cells. Northern analysis demonstrated that ZmTIP1 is expressed in all plant organs. In situ hybridization showed a high ZmTIP1 expression in meristems and zones of cell enlargement: tips of primary and lateral roots, leaf primordia, and male and female inflorescence meristems. The high ZmTIP1 expression in meristems and expanding cells suggests that ZmTIP1 is needed (a) for vacuole biogenesis and (b) to support the rapid influx of water into vacuoles during cell expansion.

Herbicide Safener-Binding Protein of Maize1 : Purification, Cloning, and Expression of an Encoding cDNA

Dichloroacetamide safeners protect maize (Zea mays L.) against injury from chloroacetanilide and thiocarbamate herbicides. Etiolated maize seedlings have a high-affinity cytosolic-binding site for the safener [3H](R,S)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazol-idine ([3H]Saf), and this safener-binding activity (SafBA) is competitively inhibited by the herbicides. The safener-binding protein (SafBP), purified to homogeneity, has a relative molecular weight of 39,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 5.5. Antiserum raised against purified SafBP specifically recognizes a 39-kD protein in etiolated maize and sorghum (Sorghum bicolor L.), which have SafBA, but not in etiolated wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), or Arabidopsis, which lack SafBA. SafBP is most abundant in the coleoptile and scarcest in the leaves, consistent with the distribution of SafBA. SBP1, a cDNA encoding SafBP, was cloned using polymerase chain reaction primers based on purified proteolytic peptides. Extracts of Escherichia coli cells expressing SBP1 have strong [3H]Saf binding, which, like binding to the native maize protein, is competitively inhibited by the safener dichlormid and the herbicides S-ethyl dipropylthiocarbamate, alachlor, and metolachlor. SBP1 is predicted to encode a phenolic O-methyltransferase, but SafBP does not O-methylate catechol or caffeic acid. The acquisition of its encoding gene opens experimental approaches for the evaluation of the role of SafBP in response to the relevant safeners and herbicides.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase1 : IV. Fusicoccin Induces the Association between the Plasma Membrane H+-ATPase and the Fusicoccin Receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H+-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H+-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H+-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H+-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H+-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H+-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H+-ATPase. Analysis of the activation state of PM H+-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.