Inhibition of granulocytic differentiation by mNotch1

Effective hematopoiesis requires the commitment of pluripotent and multipotent stem cells to distinct differentiation pathways, proliferation and maturation of cells in the various lineages, and preservation of pluripotent progenitors to provide continuous renewal of mature blood cells. While the importance of positive and negative cytokines in regulating proliferation and maturation of hematopoietic cells has been well documented, the factors and molecular processes involved in lineage commitment and self-renewal of multipotent progenitors have not yet been defined. In other developmental systems, cellular interactions mediated by members of the Notch gene family have been shown to influence cell fate determination by multipotent progenitors. We previously described the expression of the human Notch1 homolog, TAN-1, in immature hematopoietic precursors. We now demonstrate that constitutive expression of the activated intracellular domain of mouse Notch1 in 32D myeloid progenitors inhibits granulocytic differentiation and permits expansion of undifferentiated cells, findings consistent with the known function of Notch in other systems.

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

tRNA binding to the ribosomal P site is dependent not only on correct codon–anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitution–interference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNAPhe from Escherichia coli which are required for P-site binding. Stereo-specific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp- or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNAPhe and E. coli tRNAfMet indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification–interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNAPhe, we propose that specific Rp-phosphate oxygens are required for anticodon loop (“U-turn”) stabilization or are involved in interactions with the ribosome on correct tRNA–mRNA complex formation.

Nuclear tRNA aminoacylation and its role in nuclear export of endogenous tRNAs in Saccharomyces cerevisiae

Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensuring nuclear export of functional tRNAs [Lund, E. & Dahlberg, J. E. (1998) Science 282, 2082–2085]. Herein, it is documented that tRNA aminoacylation also occurs in yeast nuclei and is important for tRNA export. We propose that tRNA aminoacylation functions in one of at least two parallel paths of tRNA export in yeast. Alteration of one aminoacyl-tRNA synthetase affects export of only cognate tRNA, whereas alterations of two other aminoacyl-tRNA synthetases affect export of both cognate and noncognate tRNAs. Saturation of tRNA export pathway is a possible explanation of this phenomenon.

Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow

We have identified a rare (≈0.05–0.1%) population of cells (Thy-1hiCD16+CD44hiCD2−TCRαβ−B220−Mac-1−NK1.1−) in the adult mouse bone marrow that generates CD4+ and CD8+ TCRαβ+ T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2−CD16+CD44hiTCRαβ− to CD2+CD16int/−CD44int/−TCRαβ− cells, and a later transition to CD4+CD8+TCRαβ+ double-positive T cells that rapidly generate the CD4+ and CD8+ single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2+TCRαβ− stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.

Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.

The Optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.

The catalytic sites of 20S proteasomes and their role in subunit maturation: A mutational and crystallographic study

We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation. β1(Pre3), β2(Pup1), and β5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively. The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis. The propeptides of β6(Pre7) and β7(Pre4) are intermediately processed to their final forms by β2(Pup1) in the wild-type enzyme and by β5(Pre2) and β1(Pre3) in the β2(Pup1) inactive mutants. A role of the propeptide of β1(Pre3) is to prevent acetylation and thereby inactivation. A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits β3(Pup3), β6(Pre7), and β7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.

Homotypic Fusion of Immature Secretory Granules During Maturation Requires Syntaxin 6

Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroendocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) molecules are involved in the homotypic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocytosis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion. Subcellular fractionation studies and indirect immunofluorescence microscopy show that syntaxin 6 is sorted away during the maturation of ISGs to MSGs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SNAP-29/GS32, we could not find evidence that these target (t)-SNARE molecules are involved in homotypic ISG fusion. Nor could we find any involvement for the vesicle (v)-SNARE VAMP4, which is known to be associated with syntaxin 6. Importantly, we have shown that homotypic fusion requires the function of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t–t-SNARE interactions, either direct or indirect, may be required during fusion of ISG membranes.

Adenovirus-mediated gene delivery into neuronal precursors of the adult mouse brain.

Precursor cells found in the subventricular zone (SVZ) of the adult brain can undergo cell division and migrate long distances before differentiating into mature neurons. We have investigated the possibility of introducing genes stably into this population of cells. Replication-defective adenoviruses were injected into the SVZ of the lateral ventricle of adult mice. The adenoviruses carried a cDNA for the LacZ reporter or the human p75 neurotrophin receptor, for which species-specific antibodies are available. Injection of the viruses into the SVZ led to efficient labeling of neuronal precursors. Two months after viral injection, infected cells were detected in the olfactory bulb, a significant distance from the site of injection. Labeled periglomerular and granular neurons with extensive dendritic arborization were found in the olfactory bulb. These results demonstrate that foreign genes can be efficiently introduced into neuronal precursor cells. Furthermore, adenovirus-directed infection can lead to long-term stable gene expression in progenitor cells found in the adult central nervous system.

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.

Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.

We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process.

A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.

Nuclear-encoded proteins targeted to the chloroplast are typically synthesized with N-terminal transit peptides which are proteolytically removed upon import. Structurally related proteins of 145 and 143 kDa copurify with a soluble chloroplast processing enzyme (CPE) that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein and have been implicated in the maturation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and acyl carrier protein. The 145- and 143-kDa proteins have not been found as a heterodimer and thus may represent functionally independent isoforms encoded by separate genes. Here we describe the primary structure of a 140-kDa polypeptide encoded by cDNAs isolated by using antibodies raised against the 145/143-kDa doublet. The 140-kDa polypeptide contains a transit peptide, and strikingly, a His-Xaa-Xaa-Glu-His zinc-binding motif that is conserved in a recently recognized family of metalloendopeptidases, which includes Escherichia coli protease III, insulin-degrading enzyme, and subunit beta of the mitochondrial processing peptidase. Identity of 25-30%, concentrated near the N terminus of the 140-kDa polypeptide, is found with these proteases. Expression of CPE in leaves is not light dependent. Indeed, transcripts are present in dark-grown plants, and the 145/143-kDa doublet and proteolytic activity are both found in etioplasts, as well as in root plastids. Thus, CPE appears to be a necessary component of the import machinery in photosynthetic and nonphotosynthetic tissues, and it may function as a general stromal processing peptidase in plastids.

Macrophage-colony-stimulating factor regulates expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 by murine bone marrow macrophages.

We observed that when monocyte/macrophage precursors derived from murine bone marrow were treated with macrophage-colony-stimulating factor (M-CSF), there was a dose-dependent increase in both the number of adherent cells and the degree to which the cells were highly spread. Attachment was supported by fibronectin, but not by vitronectin or laminin, suggesting that the integrins alpha 4 beta 1 and/or alpha 5 beta 1 might mediate this event. Binding to fibronectin was blocked partially by antibodies to either integrin, and inhibition was almost complete when the antibodies were used in combination. By a combination of surface labeling with 125I and metabolic labeling with [35S]methionine and [35S]cysteine, we demonstrated that M-CSF treatment led to increased synthesis and surface expression of the two beta 1 integrins. Since attachment to fibronectin and/or stromal cells plays an important role in the maturation of other hematopoietic lineages, we propose that the action of M-CSF in the differentiation of immature monocytes/macrophages includes stimulated expression of the integrins alpha 4 beta 1 and alpha 5 beta 1, leading to interactions with components of the marrow microenvironment necessary for cell maturation.

Formation of mesostructured titania thin films using iospropoxide precursors

Mesostructured titania thin films were prepared by an evaporation-induced self-assembly process. The highly acidic sot precursors contained titanium(IV) tetraisopropoxide (TTIP) as a titanium source, a tri-block copolymer Pluronic P123 as a template, and acetylacetonate and HCl as hydrolysis inhibitors. Characteristics of the resultant titania thin films were studied using X-ray diffraction (XRD) analysis, N-2-adsorption/desorption analysis, and transmission electron microscopy (TEM). XRD and TEM investigations on the as-synthesised films revealed the appearance of cubic-like, pseudohexagonal, and lamellar mesophases; depending on the amount of water in the sols of film precursors. Template removal by a calcination process yields high surface area (320-360 m(2)/g) mesoporous materials with crystalline anatase frameworks. Water content also influences the degree of anatase crystallinity of the calcined films. Higher water content resulted in improved anatase crystallinity. These nanostructured materials are of interest for photocatalysts, pbotoelectrochemical solar cells and other photonic devices. (C) 2003 Elsevier B.V. All rights reserved.