Biomedical subjects

K Khalili

Publications and source records attributed to K Khalili.

168 records · Page 10Linked to original sources

PubMed1984

Actin mRNAs in HeLa cells. Stabilization after adenovirus infection.

We have carried out a comparative analysis of the expression of the actin genes in HeLa and adenovirus-infected HeLa cells. The rate of actin gene transcription was examined in these cells by pulse-labeling of the newly synthesized RNA and/or by in vitro transcription in nuclei isolated from uninfected or infected HeLa cells. In addition, accumulation of actin-specific heterogeneous nuclear RNA, and rate of appearance of the actin mRNAs in the cytoplasm were examined by dot and Northern blot analysis. The rate of actin gene transcription remained constant after infection of HeLa cells with adenovirus serotype 2, while the level of the actin precursor in the nuclei was slightly reduced. In the infected cells, newly synthesized actin mRNA enters the cytoplasm at a very reduced rate. The deficiency of transport does not affect the steady-state level of the messages in the cytoplasm. The half-life of cytoplasmic actin mRNAs was analyzed by traditional pulse-chase experiments and by a novel procedure using 5-6-diCl-1-beta-d-ribofuranosylbenzimidazole, which does not rely on labeled RNA. Both procedures gave identical results. Uninfected HeLa cells have actin mRNAs with relatively short half-lives, from less than six to 12 hours. In contrast, the half-lives of the actin-specific mRNAs, in the cytoplasm of adenovirus-infected cells, is greater than 14 to 24 hours. These observations suggest that, although the rate of transport of actin mRNAs to the cytoplasm is reduced upon infection with adenovirus, increased half-lives result in accumulation of actin mRNAs to normal levels in the cytoplasm.

Actins↗

PubMed1984

Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells.

Adenovirus produces a dramatic shut-off of host protein synthesis after infection of HeLa cells. The level of actin messenger RNAs remained relatively unchanged after viral infection, when assayed by in vitro translation and two-dimensional gel electrophoresis analysis of the proteins or hybridization of the total cytoplasmic RNAs to the human actin gene. The distribution of actin mRNA in the polyribosomes is altered after adenovirus infection, with small polyribosomes and monoribosomes of the infected cells occupied by actin messages untranslatable in a rabbit reticulocyte lysate. The large polyribosomes still retain enough functional mRNAs to provide significant levels of actin protein in a rabbit reticulocyte in vitro translation system. In contrast, in homologous infected cell lysates, the translation of exogenous actin mRNA is greatly reduced when compared to uninfected HeLa cell lysates. In nuclease-treated uninfected or infected HeLa cell-free extracts, translation of viral mRNA is equally efficient and higher than that of actin mRNA. Thus, translational regulatory mechanisms which include inactivation of a part of the actin mRNA population accompanied by displacement to small polysomes and/or virus-induced modification of the cellular translational machinery to discriminate against cellular actin mRNA seem to account for the sharp reduction in actin protein synthesis of adenovirus-infected cells.

Actins↗

PubMedDate not supplied

Single-stranded nucleic acid-binding protein, Pur alpha, interacts with RNA homologous to 18S ribosomal RNA and inhibits translation in vitro.

Pur alpha is a highly conserved, eukaryotic sequence-specific DNA- and RNA-binding protein involved in diverse cellular and viral functions including transcription, replication, and cell growth. Pur alpha exerts its activity in part by interacting with other viral and cellular proteins. One such protein is the human immunodeficiency virus (HIV) type I regulatory protein Tat. Earlier studies have demonstrated that this interaction is mediated by Pur alpha-associated RNA (PARNA) and that RNA immunopurified from mammalian expressed Pur alpha was capable of reconstituting the interaction between these two proteins. In the current study, we characterize four RNA species which were immunopurified with Pur alpha. Northern blot analysis with one of the PARNAs revealed a highly abundant signal of approximately 2.0 kilobases (kb) present in all cell lines tested. Sequence analysis of each of the four PARNA clones revealed a high homology to different regions of the human 18S ribosomal RNA sequence. Based on this homology, we investigated the influence of Pur alpha on translation. Luciferase assays were performed after coupled in vitro transcription/translation reactions with a vector containing a luciferase reporter construct and increasing concentrations of BSA, GST, and GST-Pur alpha. Inclusion of GST-Pur alpha in these reactions resulted in a dose-dependent inhibition of luciferase activity. Similar inhibition was observed with in vitro translation reactions performed with in vitro transcribed luciferase RNA and increasing concentrations of GST-Pur alpha. In control experiments, inclusion of increasing concentrations of GST-Pur alpha with luciferase protein resulted in no effect on luciferase activity. Taken together, these data demonstrate that Pur alpha inhibits translation reactions in vitro. Moreover, this Pur alpha-mediated inhibition of translation can be abrogated by HIV-1 Tat protein.

Animals↗

PubMedDate not supplied

In vivo validation of tissue segmentation based on a 3D feature map using both a hamster brain tumor model and stereotactically guided biopsy of brain tumors in man.

The purpose of this study was to validate our MR tissue segmentation technique using a hamster brain tumor model and malignant brain tumors in man. We used a multispectral tissue segmentation analysis. Three sets of MRI data were included: proton density, T2-weighted fast spin echo, and T1-weighted spin echo, as inputs. Three image preprocessing steps included correcting image nonuniformity, application of an anisotropic diffusion type filter, and data point selection by a qualified observer. We used the k-Nearest Neighbor segmentation algorithm, which does not require prior knowledge of the sample distribution. This choice allowed us to optimize the different tissue clusters present in three-dimensional (3D) feature space. In vivo validation of the technique was performed in hamsters harboring tumors induced with JC virus-transformed HJC-15 cells, as compared to three control animals. Human brain tumors obtained by stereotactically guided biopsy in six patients were also included in the study. Finally, brain tumors were removed from two patients who underwent conventional craniotomy using segmentation-derived images as a guide. In the hamsters, 10 tissues were correctly identified by segmentation and were confirmed histologically (P < .02). In the patients, there was also a strong correlation between our segmentation results and the tissue obtained by stereotactic biopsy (P < .01). In one of the two patients who underwent open craniotomy, segmentation images were useful in revealing tumor spread into vital areas of the brain (motor area). In conclusion, the results of segmentation correlate well with the tissues in vivo and thus warrant further clinical utilization and evaluation.

Adult↗

PubMedDate not supplied

Isolation and characterization of human actin genes cloned in phage lambda vectors.

Using Drosophila and chicken actin probes, we have selected 14 human actin lambda recombinants from a genomic library. We present a restriction maps indicating the positions of the sequences homologous to actin and to an Alu probe. Restriction mapping has revealed that nine out of ten of these clones are distinct, indicating that actin is a multigene family. Hybrid elution of HeLa cell mRNA from filters containing the recombinant DNA, followed by in vitro translation and immunoprecipitation, as well as one- or two-dimensional protein analysis, shows that these recombinants code for actin. Hybridization back to human DNA digested with restriction enzymes shows that the EcoRI fragments of at least one of the lambda recombinants (lambda HA-5) result in similar-sized human DNA fragments in the intact genome. In nuclei, a 4.5-kb mRNA precursor to the cytoplasmic 1.9-kb mRNA can be detected by hybridization with genomic or cDNA probes, indicating the presence of additional sequences and RNA processing.

Actins↗

PubMedDate not supplied

HIV-1 regulatory protein tat induces RNA binding proteins in central nervous system cells that associate with the viral trans-acting-response regulatory motif.

OBJECTIVES: To investigate some of the cellular consequences of HIV-1 Tat expression in human astrocytic cells. This study is based on evidence that cellular factors play a critical role in facilitating transcriptional activation by Tat through its interaction with the trans-acting-response (TAR) RNA element and upstream HIV-1 long terminal repeat (LTR) promoter binding site. STUDY DESIGN-METHODS: Using the previously established astrocytic cell line of human origin stably transfected with Tat cDNA, we analyzed the formation of a nucleoprotein complex consisting of three cellular proteins associated with TAR RNA using ultraviolet (UV) crosslinking and glutathione-S-transferase (GST) pull-down assays. RESULTS: UV crosslinking experiments reveal that the molecular masses of the proteins range from 50 to 62 kd. Transient transfection studies demonstrate that the presence of these proteins correlates with the ability of Tat to transactivate the HIV-1 LTR in the absence of the trinucleotide bulge, a region within TAR that has been shown to be important for Tat-TAR interaction. A combination of GST pull-down assays and RNA binding studies demonstrates that the 50-kd protein interacts with both Tat and TAR and is likely to be NF-kappa B p50. CONCLUSIONS: Taken together, these data suggest that in the absence of a functional Tat binding site such as TAR (which tethers the viral protein to the RNA), cellular protein NF-kappa B p50 may be able to bring Tat into the RNA binding complex Tat has been shown to activate expression of a variety of cellular genes that may not contain a binding site for Tat but do contain binding sites for NF-kappa B family members. The results presented in this study may be relevant for Tat-mediated transactivation of cellular as well as viral genes, both of which might contribute to the central nervous system damage associated with HIV-1 infection.

Astrocytes↗