Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.

Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant

Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515–518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells

An Fcα receptor probe of human origin was used to identify novel members of the Ig gene superfamily in mice. Paired Ig-like receptors, named PIR-A and PIR-B, are predicted from sequence analysis of the cDNAs isolated from a mouse splenic library. Both type I transmembrane proteins possess similar ectodomains with six Ig-like loops, but have different transmembrane and cytoplasmic regions. The predicted PIR-A protein has a short cytoplasmic tail and a charged Arg residue in the transmembrane region that, by analogy with the FcαR relative, suggests the potential for association with an additional transmembrane protein to form a signal transducing unit. In contrast, the PIR-B protein has an uncharged transmembrane region and a long cytoplasmic tail containing four potential immunoreceptor tyrosine-based inhibitory motifs. These features are shared by the related killer inhibitory receptors. PIR-A proteins appear to be highly variable, in that predicted peptide sequences differ for seven randomly selected PIR-A clones, whereas PIR-B cDNA clones are invariant. Southern blot analysis with PIR-B and PIR-A-specific probes suggests only one PIR-B gene and multiple PIR-A genes. The PIR-A and PIR-B genes are expressed in B lymphocytes and myeloid lineage cells, wherein both are expressed simultaneously. The characteristics of the highly-conserved PIR-A and PIR-B genes and their coordinate cellular expression suggest a potential regulatory role in humoral, inflammatory, and allergic responses.

Chaperonin filaments: The archaeal cytoskeleton?

Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea.

Constitutive tyrosine phosphorylation of the inhibitory paired Ig-like receptor PIR-B

PIR-A and PIR-B are activating and inhibitory Ig-like receptors on murine B lymphocytes, dendritic cells, and myeloid-lineage cells. The inhibitory function of PIR-B is mediated via its cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, whereas PIR-A pairs with the Fc receptor common γ chain to form an activating receptor complex. In these studies, we observed constitutive tyrosine phosphorylation of PIR-B molecules on macrophages and B lymphocytes, irrespective of the cell activation status. Splenocyte PIR-B molecules were constitutively associated with the SHP-1 protein tyrosine phosphatase and Lyn protein tyrosine kinase. In Lyn-deficient mice, PIR-B tyrosine phosphorylation was greatly reduced. Unexpectedly, tyrosine phosphorylation of PIR-B was not observed in most myeloid and B cell lines but could be induced by ligation of the PIR molecules. Finally, the phosphorylation status of PIR-B was significantly reduced in MHC class I-deficient mice, although not in mice deficient in TAP1 or MHC class II expression. These findings suggest a physiological inhibitory role for PIR-B that is regulated by endogenous MHC class I-like ligands.

A system for functional analysis of Ebola virus glycoprotein

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVΔG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVΔG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVΔG* complemented with VSV G protein (VSVΔG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVΔG*-ResGP but not to VSVΔG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role of ste9+ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10ts, cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase.

Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the “A” fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.

Terreic acid, a quinone epoxide inhibitor of Bruton’s tyrosine kinase

Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors.

Paired Ig-like receptor homologs in birds and mammals share a common ancestor with mammalian Fc receptors

Paired Ig-like receptors (PIR) that can reciprocally modulate cellular activation have been described in mammals. In the present study, we searched expressed sequence tag databases for PIR relatives to identify chicken expressed sequence tags predictive of ≈25% amino acid identity to mouse PIR. Rapid amplification of cDNA ends (RACE)-PCR extension of expressed sequence-tag sequences using chicken splenic cDNA as a template yielded two distinct cDNAs, the sequence analysis of which predicted protein products with related extracellular Ig-like domains. Chicken Ig-like receptor (CHIR)-A was characterized by its transmembrane segment with a positively charged histidine residue and short cytoplasmic tail, thereby identifying CHIR-A as a candidate-activating receptor. Conversely, CHIR-B was characterized by its nonpolar transmembrane segment and cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motifs, indicating that it may serve as an inhibitory receptor. The use of CHIR amino acid sequences in a search for other PIR relatives led to the recognition of mammalian Fc receptors as distantly related genes. Comparative analyses based on amino acid sequences and three-dimensional protein structures provided molecular evidence for common ancestry of the PIR and Fc receptor gene families.

Regulation of c-maf gene expression by Pax6 in cultured cells

c-Maf is a bZip transcription factor expressed in developmental and cellular differentiation processes. Recently, a c-maf knockout mouse model, showing abnormal lens development, has been reported. In order to study the regulation mechanisms of c-maf gene expression during the differentiation process we have cloned and functionally characterized the rat c-maf (maf-2) gene. The rat c-maf gene is an intronless gene, covering a length of 3.5 kb. Transient transfection analysis of the 5′-flanking region of the c-maf gene using luciferase as the reporter gene shows that Pax6, a master transcription factor for lens development, strongly activates the c-maf promoter construct. Endogenous c-maf is also activated by the Pax6 expression vector. Electrophoresis mobility shift assay and DNase I footprinting analysis show that at least three Pax6-binding sites are located in the 5′-flanking and 5′-non-coding regions of the rat c-maf gene. The c-maf gene was also markedly activated by its own product, c-Maf, through the MARE (Maf recognition element), suggesting that a positive autoregulatory mechanism controls this gene. In situ hybridization histochemical detection of Pax6 and c-Maf in the E14 lens showed that both mRNAs are expressed in the lens equator where lens epithelial cells are differentiating to lens fiber cells. These results suggest that a Pax6/c-Maf transcription factor cascade is working in lens development.

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen

Serological expression cloning of antigens eliciting a humoral immune response to a syngeneic mouse sarcoma identified pem (mouse placenta and embryonic expression gene) as a new member of the cancer/testis family. To identify the human homologue of pem, mouse pem sequences and pem-related expressed sequence tags from human testis were used as PCR primers for amplification using human testis cDNA. However, rather than pem, another gene, designated OY-TES-1, was isolated and found to be the human homologue of proacrosin binding protein sp32 precursor originally identified in mouse, guinea pig, and pig. OY-TES-1 maps to chromosome 12p12-p13 and contains 10 exons. Southern blot analysis suggests the presence of two OY-TES-1-related genes in the human genome. In normal tissues, OY-TES-1 mRNA was expressed only in testis, whereas in malignant tissues, a variable proportion of a wide array of cancers, including bladder, breast, lung, liver, and colon cancers, expressed OY-TES-1. Serological survey of 362 cancer patients with a range of different cancers showed antibody to OY-TES-1 in 25 patients. No OY-TES-1 sera reactivity was found in 20 normal individuals. These findings indicate that OY-TES-1 is an additional member of the cancer/testis family of antigens and that OY-TES-1 is immunogenic in humans.

Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in the Biosynthesis of Phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.

Formate Dehydrogenase, an Enzyme of Anaerobic Metabolism, Is Induced by Iron Deficiency in Barley Roots1

To identify the proteins induced by Fe deficiency, we have compared the proteins of Fe-sufficient and Fe-deficient barley (Hordeum vulgare L.) roots by two-dimensional polyacrylamide gel electrophoresis. Peptide sequence analysis of induced proteins revealed that formate dehydrogenase (FDH), adenine phosphoribosyltransferase, and the Ids3 gene product (for Fe deficiency-specific) increased in Fe-deficient roots. FDH enzyme activity was detected in Fe-deficient roots but not in Fe-sufficient roots. A cDNA encoding FDH (Fdh) was cloned and sequenced. Fdh expression was induced by Fe deficiency. Fdh was also expressed under anaerobic stress and its expression was more rapid than that induced by Fe deficiency. Thus, the expression of Fdh observed in Fe-deficient barley roots appeared to be a secondary effect caused by oxygen deficiency in Fe-deficient plants.