The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing.

Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders.

Structure and assembly of immature HIV.

The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled immature Gag lattice to approximately 17-A resolution. Gag is arranged in the immature virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein-protein interfaces involved in Gag assembly, and to identify the structural transformations associated with virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped viruses of variable sizes.

Prospective study of cyclin D1 overexpression in Barrett's esophagus: association with increased risk of adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma commonly arises from a precancerous condition, Barrett's esophagus, in which the normal squamous epithelium is replaced by a columnar cell-lined epithelium. Genetic alterations occurring in this process could serve as biomarkers for the risk of malignant progression, improve surveillance, and contribute to early diagnosis. We examined two potential biomarkers, cyclin D1 and p53, in a prospective cohort of Barrett's esophagus patients. METHODS: A total of 307 patients were enrolled in an endoscopic surveillance cohort, and esophageal biopsy specimens were collected at each endoscopy. Incident cases of adenocarcinoma were matched to control patients within the cohort by duration of follow-up, age, sex, and length of columnar cell-lined epithelium at recruitment. Biopsy specimens were analyzed for cyclin D1 and p53 protein levels by immunohistochemistry. Statistical tests were two-sided. RESULTS: A total of 12 cases of adenocarcinoma occurred within the follow-up period, and tumor biopsy specimens from 11 cases stained positive for cyclin D1. Biopsy specimens from eight of these patients taken at recruitment also stained positive for cyclin D1. A case-control analysis of biopsy specimens obtained at recruitment revealed a statistically significantly increased risk of progression to adenocarcinoma in Barrett's esophagus patients whose biopsy specimens were cyclin D1 positive (odds ratio [OR] = 6. 85; 95% confidence interval [CI] = 1.57-29.91; P =.0106) but not in patients whose biopsy specimens were p53 positive (OR = 2.99; 95% CI = 0.57-15.76; P =.197). CONCLUSIONS: Cyclin D1-positive staining could be a useful biomarker in identifying Barrett's esophagus patients at high risk of esophageal adenocarcinoma. Given the complexity of genetic alterations in the natural history of this cancer, additional biomarkers will be required to increase the sensitivity and specificity of molecular diagnosis.

Characterization of chromium effects on a rat liver epithelial cell line and their relevance to in vitro transformation.

Chronic exposure to low concentrations or brief exposures to high concentrations of hexavalent chromium (K2CrO4) transformed a rat liver epithelial cell line as assessed by anchorage-independent growth. A clone of the transformed cells produced tumors in syngeneic animals, all of which were determined to be high grade carcinomas. The effects of various doses of chromium on cytotoxicity and cell cycle were established and related to ultimate numbers of transformants in the population. Prior to the onset of cytotoxicity, linear rates of uptake were observed at different external concentrations of chromium and the total intracellular level increased with increasing concentration. This may be due to competition for transport. A plateau in level of chromium accumulation after prolonged exposure to a low concentration (10 microM) of chromium observed with the eventual return to normal growth provided evidence for the induction of a protective mechanism. In addition, cells surviving prolonged treatment with low concentrations of chromium became resistant to the cytotoxic effects of high concentrations of the metal, further suggesting that the cells become adapted to the presence of chromium. An increase in the amount of protein associated with extracted nucleic acids was detected at the optimal transforming dose and this did not correlate with cytotoxic effects. The effectiveness of chromium in transforming the adult rat liver epithelial cell line may depend on the intracellular level of accumulation, the rate of chromium uptake, and the ability of the cell to activate a protective mechanism. The initiation of stable nucleic acid-protein complexes observed under the optimal conditions for transformation may be associated with an inability of the cell to activate a protective mechanism rapidly enough to prevent effects on the nucleus at a high concentration (1 mM) of chromium.

Lectin-binding proteins in nuclear preparations from rat liver and malignant tumors.

Lectin binding [concanavalin A, biotinylated ricinus communis agglutinin, and biotinylated succinylated wheat germ agglutinin (B-SWGA)] was used to detect the glycosylated proteins associated with a residual protein fraction [insoluble in 4% sodium dodecyl sulfate and termed the nuclear residual fraction (NRF)] or with nuclear matrix preparations from normal rat liver, azo dye (3'-MeDAB)-induced rat hepatoma, and Walker 256 transplantable carcinosarcoma. One- and two-dimensional gel electrophoresis were used with lectins, polyclonal antisera, and monoclonal antibody binding to characterize some of the glycoconjugates. Two polypeptide bands with approximate molecular weights of 95,000 and 55,000, shown previously to be present only in the induced tumor cells and the Walker 256 tumor, were reactive with lectins. In addition, a Mr 62,000 protein reacted only with B-SWGA in the nuclear matrix fractions from normal rat liver and the induced hepatoma. A polypeptide band (approximate molecular weight, 213,000) in the Walker 256 NRF reacted with concanavalin A and biotinylated ricinus communis agglutinin. One polypeptide band (approximate molecular weight, 182,000) reacted with concanavalin A in all three tissues, with biotinylated ricinus communis agglutinin and B-SWGA in the Walker NRF, and with B-SWGA in the hepatoma NRF. Another polypeptide band (approximate molecular weight, 138,000), reactive with all three lectins, was present in all three tissues. Our findings are consistent with previous reports of lectin binding proteins in the eukaryotic cell nucleus and indicate that certain glycoproteins isolated in nuclear preparations are found specifically in 3'-MeDAB-induced hepatoma and Walker 256 transplantable carcinosarcoma.

cis-diamminedichloroplatinum(II) cross-linking of the human myeloid cell nuclear differentiation antigen to DNA in HL-60 cells following 1,25-dihydroxy vitamin D3-induced monocyte differentiation.

Protein-DNA interactions of the human myeloid cell nuclear differentiation antigen (MNDA) were examined in vivo in proliferating HL-60 promyelocytic leukemia cells and following induction of differentiation by 1,25-dehydroxyvitamin D3. Intact cells were treated with the reversible cross-linking agent cis-diamminedichloroplatinum(II) and the MNDA levels in the isolated protein-DNA complexes were determined. Less than 1% of the total intracellular level of MNDA was cross-linked to DNA in the noninduced proliferating HL-60 cells. Once the cells were induced to differentiate into monocytes, the amount of antigen cross-linked to the DNA increased to over 5% of the total intracellular level. The increased efficiency of cross-linking the MNDA to DNA was specific for monocyte-induced HL-60 differentiation, achieved with three inducers, and was not observed in association with granulocyte-induced differentiation. On a molar basis the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) was the most effective inducer of monocyte differentiation, followed by 1,25-dihydroxy-16-ene-23-ynecholecalciferol which was more effective than 1,25-dihydroxycholecalciferol. A cesium chloride gradient analysis of the nucleic acid-protein fraction isolation from cis-diamminedichloroplatinum(II)-treated, monocyte-induced HL-60 cells documented the authenticity of the association between the MNDA and DNA. The results indicate that a significant level of chromatin reorganization may accompany monocyte-induced differentiation that leads to much higher levels of MNDA-DNA cross-linking to DNA. The expression of the MNDA is restricted to human myeloid cells and the present results indicate that a fraction of this low abundance nuclear protein is specifically located near the DNA [within cis-diamminedichloroplatinum(II) cross-linking distance] and that this association may be modulated specifically during monocyte differentiation.

Cryo-Electron Tomography and Subtomogram Averaging.

Cryo-electron tomography (cryo-ET) allows 3D volumes to be reconstructed from a set of 2D projection images of a tilted biological sample. It allows densities to be resolved in 3D that would otherwise overlap in 2D projection images. Cryo-ET can be applied to resolve structural features in complex native environments, such as within the cell. Analogous to single-particle reconstruction in cryo-electron microscopy, structures present in multiple copies within tomograms can be extracted, aligned, and averaged, thus increasing the signal-to-noise ratio and resolution. This reconstruction approach, termed subtomogram averaging, can be used to determine protein structures in situ. It can also be applied to facilitate more conventional 2D image analysis approaches. In this chapter, we provide an introduction to cryo-ET and subtomogram averaging. We describe the overall workflow, including tomographic data collection, preprocessing, tomogram reconstruction, subtomogram alignment and averaging, classification, and postprocessing. We consider theoretical issues and practical considerations for each step in the workflow, along with descriptions of recent methodological advances and remaining limitations.

Characterization of the human myeloid cell nuclear differentiation antigen gene promoter.

MNDA (myeloid cell nuclear differentiation antigen) is an interferon alpha regulated nuclear protein expressed only in cells of the human myelomonocytic lineage. To identify mechanisms responsible for this lineage-specific and interferon-regulated expression, the 5' flanking sequence of the gene has been characterized. Two interferon-stimulated response elements (ISRE) flank a multiple transcription start site region identifying MNDA as a TATA-less interferon-regulated gene. Other DNA elements present include a cluster of Myb sites, several Ets, an Ets related PU.1 site and an Sp1 site located within 600 bp of the transcription start sites. In addition, DNA methylation was revealed as one of the possible factors in establishing MNDA expression. The 5' flanking sequence has promoter activity which is elevated by interferon alpha. The findings indicate that MNDA expression is regulated by mechanisms similar to other myelomonocytic cell specific genes and genes up-regulated by interferon alpha.

MNDA binds NPM/B23 and the NPM-MLF1 chimera generated by the t(3;5) associated with myelodysplastic syndrome and acute myeloid leukemia.

The myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in developing cells of the human myelomonocytic lineage, including the end-stage monocytes/macrophages and granulocytes. Nuclear localization, lineage- and stage-specific expression, association with chromatin, and regulation by interferon alpha indicate that this protein is involved in regulating gene expression uniquely associated with the differentiation process and/or function of the monocyte/macrophage. MNDA does not bind specific DNA sequences, but rather a set of nuclear proteins that includes nucleolin (C23). Both in vitro binding assays and co-immunoprecipitation were used to demonstrate that MNDA also binds protein B23 (nucleophosmin/NPM). Three reciprocal chromosome translocations found in certain cases of leukemia/lymphoma involve fusions with the NPM/B23 gene, t(5;17) NPM-RARalpha, t(2;5) NPM-ALK, and the t(3;5) NPM-MLF1. In the current study, MNDA was not able to bind the NPM-ALK chimera originating from the t(2;5) and containing residues 1-117 of NPM. However, MNDA did bind the NPM-MLF1 product of the t(3;5) that contains the N-terminal 175 residues of NPM. The additional 58 amino acids (amino acids 117-175) of the NPM sequence that are contained in the product of the NPM-MLF1 fusion gene relative to the product of the NPM-ALK fusion appear responsible for MNDA binding. This additional NPM sequence contains a nuclear localization signal and clusters of acidic residues believed to bind nuclear localization signals of other proteins. Whereas NPM and nucleolin are primarily localized within the nucleolus, MNDA is distributed throughout the nucleus including the nucleolus, suggesting that additional interactions define overall MNDA localization.

VESICULAR TRANSPORT. A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.

Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

MNDA dimerizes through a complex motif involving an N-terminal basic region.

Human myeloid cell nuclear differentiation antigen (MNDA) is a myelomonocytic lineage-specific protein that influences gene expression through interactions with other nuclear proteins and transcription factors. MNDA also self-associates and chemical cross-linking was used to demonstrate that MNDA forms a dimer. C-terminal and internal deletion mutants were used to identify two regions in the N-terminal half of MNDA essential for self-association. One region contains an imperfect leucine zipper and the second is highly enriched in basic residues. The sequences that are essential for dimerization are separated by a highly basic amphipathic alpha-helical region which was not required for dimerization.

The in vitro transformation of a rat liver epithelial cell line with chromium.

The transformation of a rat liver epithelial cell line under a wide range of doses of chromium was determined by anchorage-independent growth and tumor formation in syngeneic animals. Chronic exposure to low concentrations and brief exposure to high concentrations of hexavalent chromium (K2CrO4) transformed the cells, but one dose (1 mM K2CrO4, 2 h) was clearly optimal in this regard. The cytotoxicity, effects on cell cycle, rates of chromium uptake, and mutagenic activity under the different treatment conditions were evaluated. The results showed that cells could adapt to the presence of chromium under certain treatment conditions, but this was not the case for the optimal transforming dose. Cells treated with chromium above the optimal transforming dose showed evidence of a transient G2 arrest, whereas all lower levels of treatment did not. A low level continuous exposure to chromate was mutagenic, whereas high level short exposures, including the optimal transforming dose, were not. An increase in the amount of protein complexed with isolated nucleic acids was detected in cells following treatment with the optimal transforming dose of chromate. The results indicate that the effects of chromium on this in vitro system vary with dose, and the identification of those events relevant to metal carcinogenesis will require consideration of treatment conditions.

Production and use of rat monoclonal antibodies to the human myeloid cell nuclear differentiation antigen.

A dot immunoblot screening assay was used to identify rat monoclonal antibodies to a human myeloid cell differentiation-specific nuclear antigen (MNDA). The selection was based on the positive reaction of hybridoma cell supernatants with a concentrated nuclear protein extract prepared from late stage human myeloid leukemia cells that express MNDA (HL-60) coincident with a negative reaction with the same extract prepared from a non-expressing more immature human myeloid leukemia cell line. The approach provided an efficient method for obtaining monoclonal antibodies to a specific low abundance nuclear antigen that has not been purified. Sixteen wells from three fusions contained antibody displaying a specific reaction with the nuclear protein fraction obtained from the HL-60 cells. Immunoblotting analysis revealed that all of the sixteen specific hybridoma cell lines produced antibody against the same Mr 55,000 nuclear antigen. Selecting hybridoma cells that produce antibody reactive with the native antigen provided antibody suitable for detecting MNDA in immunocytochemical tests. The rat monoclonal antibodies were purified and coupled to CNBr-activated agarose and carbonyldiimidazole-activated agarose. Although both antibody affinity matrices exhibited the same antigen binding capacities, the matrix prepared using carbonyldiimidazole-activated agarose bound the MNDA with a high level of specificity while the matrix prepared from CNBr-activated agarose bound numerous other nuclear proteins.

A new method to model membrane protein structure based on silent amino acid substitutions.

The importance of accurately modeling membrane proteins cannot be overstated, in lieu of the difficulties in solving their structures experimentally. Often, however, modeling procedures (e.g., global searching molecular dynamics) generate several possible candidates rather then pointing to a single model. Herein we present a new approach to select among candidate models based on the general hypothesis that silent amino acid substitutions, present in variants identified from evolutionary conservation data or mutagenesis analysis, do not affect the stability of a native structure but may destabilize the non-native structures also found. The proof of this hypothesis has been tested on the alpha-helical transmembrane domains of two homodimers, human glycophorin A and human CD3-zeta, a component of the T-cell receptor. For both proteins, only one structure was identified using all the variants. For glycophorin A, this structure is virtually identical to the structure determined experimentally by NMR. We present a model for the transmembrane domain of CD3-zeta that is consistent with predictions based on mutagenesis, homology modeling, and the presence of a disulfide bond. Our experiments suggest that this method allows the prediction of transmembrane domain structure based only on widely available evolutionary conservation data.

Characterization of the human myeloid cell nuclear differentiation antigen: relationship to interferon-inducible proteins.

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed specifically in cells of the granulocyte/monocyte lineage. The MNDA has been isolated by using a monoclonal antibody affinity matrix and reversed-phase high performance liquid chromatography. Its NH2-terminal sequence has been obtained, as well as additional sequence information derived from peptides produced by cyanogen bromide and SV8 protease cleavages. Meaningful similarities were observed in extended regions between the MNDA and the reported beta interferon-inducible proteins, 202 and 204, from Ehrlich ascites mouse tumor cells. An amphipathic, basic alpha-helical region, showing no similarity to the 202 and 204 proteins, exhibited close similarity to a region in the interferon response factor-2, a protein which binds the interferon stimulated response element. The relatively high number of S(T)PXX motifs present in the partial amino acid sequence of the MNDA, described herein, suggests that the MNDA binds DNA and is a transcription factor.

Human hematopoietic cell specific nuclear protein MNDA interacts with the multifunctional transcription factor YY1 and stimulates YY1 DNA binding.

The human myeloid nuclear differentiation antigen, MNDA, is expressed only in myelomonocytic and a subset of B lymphoid hematopoietic cells. MNDA is uniformly distributed throughout the interphase cell nucleus and associates with chromatin, but does not bind specific DNA sequences. We recently demonstrated that MNDA binds nucleolin and nucleophosmin/NPM/B23 and both of these nuclear proteins bind the ubiquitous zinc finger transcription factor YY1. Investigations of the possible effect of MNDA on the interaction between YY1 and NPM, showed that MNDA bound YY1 directly under both in vitro and in vivo conditions. The MNDA-YY1 interaction enhanced the affinity of YY1 for its target DNA and decreased its rate of dissociation. The N-terminal half (200 amino acids) of MNDA was sufficient for maximum enhancement of YY1 DNA binding and a portion of this sequence was responsible for binding YY1. MNDA participated in a ternary complex with YY1 and the YY1 target DNA element. The results show that MNDA affects the ability of YY1 to bind its target DNA sequnce and that MNDA participates in a ternary complex possibly acting as a cofactor to impart lineage specific features to YY1 function.

Cloning and expression of the human myeloid cell nuclear differentiation antigen: regulation by interferon alpha.

The human myeloid cell nuclear differentiation antigen (MNDA) is a protein of 406 amino acids that is expressed specifically in granulocytes, monocytes and earlier stage cells of these lineages. Degenerate oligonucleotides that could encode regions of MNDA amino acid sequence were used to amplify the MNDA cDNA sequence using the polymerase chain reaction. The amplified cDNA product was sequenced to confirm that it encoded the MNDA protein. It was then used as a probe to isolate five clones from a human bone marrow lambda gt10 cDNA library. A clone containing a 1,672 base pair cDNA insert was sequenced and found to encode the entire MNDA open reading frame, as well as 5' and 3' untranslated regions. The primary structure of the MNDA contains extensive regions of sequence similarity with the protein products of the interferon-inducible genes: 204 and interferon regulatory factor 2. In addition, a 12-base sequence matching the interferon-stimulated response element consensus sequence [GAAAN(N)GAAA] is located in the 5' untranslated region of the MNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that express the antigen and the level of MNDA mRNA was elevated in cells treated with either recombinant or natural interferon alpha. The MNDA mRNA was not induced by interferon alpha in cells that do not exhibit a constitutive level of the MNDA mRNA. The MNDA contains sequence motifs found in gene regulatory proteins. The expression and the primary structure of the MNDA indicates that it plays a role in the granulocyte/monocyte cell-specific response to interferon.

Comparison of chicken erythroid cell nuclear isolation methods using morphological, immunochemical and biochemical criteria.

Chicken erythroid nuclei were prepared using four published methods. Our findings indicate that nuclei prepared by nitrogen cavitation are less likely to be contaminated with plasma membrane fragments than those made by procedures involving cell disruption by hypotonic lysis. However, globin gene sequences were much less sensitive to DNase I digestion in nuclei prepared by nitrogen cavitation. This suggests that the conformation of chromatin was altered by the cavitation procedure. Analysis of the proteins solubilized during limited DNase I digestion of nuclei prepared by both hypotonic lysis and cavitation revealed no appreciable differences in HMG proteins but a notable difference in the RNP-associated proteins and core histones.

Human myeloid cell nuclear differentiation antigen binds specifically to nucleolin.

The human myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in cells of the myelomonocytic lineage and regulated by interferon alpha in a cell-specific fashion. MNDA is also a member of a family of interferon-regulated genes of unknown function. In an effort to elucidate the function of MNDA, three techniques (affinity purification, coimmunoprecipitation, and protein blot assay) were used to characterize its specific protein binding activities. Microsequence analysis showed that MNDA bound the 100 kDa nucleolin protein. The identification of nucleolin was confirmed by immunoreaction with specific antibodies. MNDA contains motifs which could account for specific binding to nucleolin. Nucleolin binds other macromolecules and exhibits features consistent with roles in signal transduction, production of ribosomes, nuclear matrix structure, and regulation of transcription. The present results indicate that the function of MNDA is most likely related to interactions with other proteins. Through these associations, MNDA could contribute cell/lineage- and differentiation-specific limits to the function of ubiquitous proteins such as nucleolin. Further analysis of MNDA protein binding could be critical to elucidating the function of MNDA and could contribute to understanding the function of the products of other members of this interferon-inducible family of genes.

Regulation and specificity of MNDA expression in monocytes, macrophages, and leukemia/B lymphoma cell lines.

The expression of the human myeloid cell nuclear differentiation antigen (MNDA) was observed specifically in cells of the granulocyte-macrophage lineage in our earlier reports. The specificity of MNDA expression for cells in the granulocyte-macrophage lineage was reexamined in cell lines established from patients with Philadelphia chromosome-positive chronic myeloid leukemia. Cell lines that expressed MNDA exhibited myeloid cell features and granulocyte or monocyte differentiation could be induced in vitro, while cell lines exhibiting properties of very early stage cells or multipotential cells did not express MNDA. Cells originating from cases of Burkitt's lymphoma were negative. By contrast, three lymphoblastoid cell lines (immortalized in vitro with Epstein-Barr virus) were weakly positive and MNDA was up-regulated by interferon-alpha (IFN-alpha) treatment. As we reported previously, MNDA mRNA level in adherent monocytes is elevated by IFN-alpha; in this study, we further assessed MNDA expression in in vitro monocyte-derived macrophages. Three additional agents (endotoxin, phytohemagglutinin, and phorbol ester) and other conditions that affect function, cytokine production, differentiation, and/or growth of monocytes were examined for their ability to alter MNDA expression. The results varied with the agent, cell type, and stage of differentiation. Changes in MNDA expression occurred slowly (hours to days), suggesting that MNDA could mediate changes realized over a long period. The results also reveal a discordance in certain MNDA positive cells between steady-state levels or changes in levels of protein and mRNA indicating that the regulation of MNDA expression occurs at more than one point. Changes in MNDA expression are consistent with a role in opposing macrophage differentiation and activation of monocytes/macrophages.