Critical role of reverse transcriptase in the inhibitory mechanism of CNI-H0294 on HIV-1 nuclear translocation.

HIV-1 replication requires the translocation of viral genome into the nucleus of a target cell. We recently reported the synthesis of an arylene bis(methyl ketone) compound (CNI-H0294) that inhibits nuclear targeting of the HIV-1 genome and thus HIV-1 replication in monocyte cultures. Here we demonstrate that CNI-H0294 inhibits nuclear targeting of HIV-1-derived preintegration complexes by inactivating the nuclear localization sequence of the HIV-1 matrix antigen in a reaction that absolutely requires reverse transcriptase. This drug/reverse transcriptase interaction defines the specificity of its antiviral effect and is most likely mediated by the pyrimidine side-chain of CNI-H0294. After binding to reverse transcriptase, the carbonyl groups of CNI-H0294 react with the nuclear localization sequence of matrix antigen and prevent its binding to karyopherin alpha, the cellular receptor for nuclear localization sequences that carries proteins into the nucleus. Our results provide a basis for the development of a novel class of compounds that inhibit nuclear translocation and that can, in principle, be modified to target specific infectious agents.

Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.

The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.

Influence of light on DNA content of Helianthus annuus Linnaeus.

Mean nuclear 2C DNA content (C equaling haploid DNA per nucleus) of the first leaf of the sunflower, Helianthus annuus L., is influenced by the quality and the quantity of light. Seedlings of two inbred lines, RHA 299 and RHA 271 were germinated and grown in controlled environmental conditions. Lighting was adjusted to provide different combinations of photon flux densities and red to far red (R:FR) ratios. At R:FR = 5.8 and photon flux densities of 170 mumol.m-2.s-1, 200 mumol.m-2.s-1, and 230 mumol.m-2.s-1, DNA content remained high and relatively constant (x = 6.97 pg for RHA 271 and x = 7.32 pg for RHA 299). When the photon flux density range (R:FR = 5.8) was elevated to 350 mumol.m-2.s-1, 410 mumol.m-2.s-1, and 470 mumol.m-2.s-1, mean DNA content was reduced to 6.23 pg (RHA 271) and 6.46 pg (RHA 299). At R:FR = 1.5, mean DNA content was consistently high (7.2-7.9 pg) only at the lowest photon flux density of 170 mumol.m-2.s-1. Significant decreases in DNA content (< or = 12%) were observed at photon flux densities of 200 mumol.m-2.s-1 and 230 mumol.m-2.s-1. At the higher photon flux densities (350 mumol.m-2.s-1, 410 mumol.m-2.s-1, and 470 mumol.m-2.s-1) and R:RF = 1.5, the plants had extremely low DNA contents (mean x = 3.36 pg for RHA 271 and 3.41 pg for RHA 299) and high between-plant variance. The instability of DNA content, particularly for plants grown under light that is far red rich, suggests that phytochromes may be involved in regulating DNA content of the sunflower.

The miniaturized nuclear genome of eukaryotic endosymbiont contains genes that overlap, genes that are cotranscribed, and the smallest known spliceosomal introns.

Chlorarachniophyte algae contain a complex, multi-membraned chloroplast derived from the endosymbiosis of a eukaryotic alga. The vestigial nucleus of the endosymbiont, called the nucleomorph, contains only three small linear chromosomes with a haploid genome size of 380 kb and is the smallest known eukaryotic genome. Nucleotide sequence data from a subtelomeric fragment of chromosome III were analyzed as a preliminary investigation of the coding capacity of this vestigial genome. Several housekeeping genes including U6 small nuclear RNA (snRNA), ribosomal proteins S4 and S13, a core protein of the spliceosome [small nuclear ribonucleoprotein (snRNP) E], and a cip-like protease (clpP) were identified. Expression of these genes was confirmed by combinations of Northern blot analysis, in situ hybridization, immunocytochemistry, and cDNA analysis. The protein-encoding genes are typically eukaryotic in overall structure and their messenger RNAs are polyadenylylated. A novel feature is the abundance of 18-, 19-, or 20-nucleotide introns; the smallest spliceosomal introns known. Two of the genes, U6 and S13, overlap while another two genes, snRNP E and clpP, are cotranscribed in a single mRNA. The overall gene organization is extraordinarily compact, making the nucleomorph a unique model for eukaryotic genomics.

Modifications of RNA processing modulate the expression of hemoglobin genes.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.

Kinetics of self-assembling microtubules: an "inverse problem" in biochemistry.

Experimental time series for a nonequilibrium reaction may in some cases contain sufficient data to determine a unique kinetic model for the reaction by a systematic mathematical analysis. As an example, a kinetic model for the self-assembly of microtubules is derived here from turbidity time series for solutions in which microtubules assemble. The model may be seen as a generalization of Oosawa's classical nucleation-polymerization model. It reproduces the experimental data with a four-stage nucleation process and a critical nucleus of 15 monomers.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Structure and evolution of the compact radio source in NGC 1275.

Investigations of the fine-scale structure in the compact nucleus of the radio source 3C 84 in NGC 1275 (New General Catalogue number) are reported. Structural monitoring observations beginning as early as 1976, and continuing to the present, revealed subluminal motions in a jet-like relatively diffuse region extending away from a flat-spectrum core. A counterjet feature was discovered in 1993, and very recent nearly simultaneous studies have detected the same feature at five frequencies ranging from 5 to 43 GHz. The counterjet exhibits a strong low-frequency cutoff, giving this region of the source an inverted spectrum. The observations are consistent with a physical model in which the cutoff arises from free-free absorption in a volume that surrounds the core but obscures only the counterjet feature. If such a model is confirmed, very-long-baseline radio interferometry observations can then be used to probe the accretion region, outside the radio jet, on parsec scales.

Subparsec-scale structure and evolution of Centaurus A (NGC5128).

We present a series of 8.4-GHz very-long-baseline radio interferometry images of the nucleus of Centaurus A (NGC5128) made with a Southern Hemisphere array, representing a 3.3-year monitoring effort. The nuclear radio jet is approximately 50 milliarcseconds in extent, or at the 3.5-megaparsec distance of NGC5128, approximately 1 parsec in length. Subluminal motion is seen and structural changes are observed on time scales shorter than 4 months. High-resolution observations at 4.8 and 8.4 GHz made in November 1992 reveal a complex morphology and allow us to unambiguously identify the self-absorbed core located at the southwestern end of the jet.

Reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to a supraphysiologic glucose concentration is associated with loss of STF-1 transcription factor expression.

Chronic exposure of HIT-T15 beta cells to elevated glucose concentrations leads to decreased insulin gene transcription. The reduction in expression is accompanied by diminished binding of a glucose-sensitive transcription factor (termed GSTF) that interacts with two (A+T)-rich elements within the 5' flanking control region of the insulin gene. In this study we examined whether GSTF corresponds to the recently cloned insulin gene transcription factor STF-1, a homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and pancreas. We found that an affinity-purified antibody recognizing STF-1 supershifted the GSTF activator complex formed from HIT-T15 extracts. In addition, we demonstrated a reduction in STF-1 mRNA and protein levels that closely correlated with the change in GSTF binding in HIT-T15 cells chronically cultured under supraphysiologic glucose concentrations. The reduction in STF-1 expression in these cells could be accounted for by a change in the rate of STF-1 gene transcription, suggesting a posttranscriptional control mechanism. In support of this hypothesis, no STF-1 mRNA accumulated in HIT-T15 cells passaged in 11.1 mM glucose. The only RNA species detected was a 6.4-kb STF-1 RNA species that hybridized with 5' and 3' STF-1-specific cDNA probes. We suggest that the 6.4-kb RNA represents an STF-1 mRNA precursor and that splicing of this RNA is defective in these cells. Overall, this study suggests that reduced expression of a key transcriptional regulatory factor, STF-1, contributes to the decrease in insulin gene transcription in HIT-T15 cells chronically cultured in supraphysiologic glucose concentration.

Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer.

We have developed methods to use anticyclin A, B, and E antibodies as reagents to specifically detect proliferating cells in specific phases of the cell cycle in formalin-fixed, paraffin-embedded sections of tissues and cells. Staining of 48 archival cases of breast cancer showed that these antibodies estimate the tumor proliferation fraction and therefore are potentially useful for the prediction of prognosis. A subset of cancers had a high frequency of tumor cells expressing cyclins A and E, out of proportion to other proliferation markers, suggesting that these tumors may have deregulated cyclin expression. In addition to recognizing authentic cyclin E in the nucleus of proliferating cells, anticyclin E antibody cross-reacted with a cytoplasmic protein in nonproliferating endothelial cells. This cross-reaction allows the simultaneous visualization and quantitation of microvessels in the tumors, measuring a second potential predictor of breast cancer prognosis, tumor angiogenesis.

(Table 8.4, Page 220-223) Chemical composition of different growth structures of manganese nodules, SONNE cruise SO78

In the nodule field of the Peru Basin, situated south of the zone of high bioproductivity, a relatively high flux of biogenic matter explains a distinct redox boundary at about 10 cm depth separating very soft oxic surface sediments from stiffer suboxic sediments. Maximum abundance (50 kg/m**2) of diagenetic nodules is found near the calcite compensation depth (CCD), currently at 4250 m. There, the accretion rate of nodules is much higher (100 mm/Ma) than on ridges (5 mm/Ma). Highest accretion rates are found at the bottom of large nodules that repeatedly sink to a level immediately above the redox boundary. There, distinct diagenetic growth conditions prevail and layers of dense laminated Mn oxide of very pure todorokite are formed. The layering of nodules is mainly the result of organisms moving nodules within the oxic surface sediment from diagenetic to hydrogenetic environments. The frequency of such movements is much higher than that of climatic changes. Two types of nodule burial occur in the Peru Basin. Large nodules are less easily moved by organisms and become buried. Consequently, buried nodules generally are larger than surface nodules. This type of burial predominates in basins. At ridges where smaller nodules prevail, burial is mainly controlled by statistical selection where some nodules are not moved up by organisms.

Analysis of Mn-deposits from Lake Eningi-Lampi

The processes of formation of iron-manganese nodules and crusts have been studied on an example of the Eningi-Lampi lake, Central Karelia, where the relationships between the source of the ore, sedimentary materials and areas of their accumulation prove relatively simple and apparent. Nodules and crusts are composed mostly by birnessite, amorphous hydrous ferric oxides and hydro-goethite. They occur, as a rule, on the surface of relatively coarse-grained sediments, at the ground-water interface. Considerably in a lesser extent are found the nodules in the upper part (0ó5 cm) of the red-brown flooded watery mud covering dark-green, black muds. The nucleus of nodules, or the basis of crusts of iron-manganese hydroxides are various, frequently altered, fragments of rocks, sometimes pieces of wood. Distribution of Mn and Fe in sediments and waters of the lake is considered. It is shown that the Mn/Fe ratio decreases considerably in waters, sediments and nodules of the lake while moving off a distance from the source. The main role in the process of formation of iron-manganese nodules belongs to the selective chemosorption interaction (with auto-catalytic oxidation) of component-bearing solutions with active surfaces.

Investigating the Effect of Histone H4 Sumoylation on Chromatin Structure and Function

Thesis (Ph.D.)--University of Washington, 2016-06