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F Wu

Publications and source records attributed to F Wu.

At least 199 records · Page 11Linked to original sources
PubMed1991

tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185.

The TAR element extending from -17 to +80 in the human immunodeficiency virus long terminal repeat (HIV LTR) is required for activation of gene expression by the tat trans-activator protein. TAR RNA forms a stable stem-loop structure, and mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and the bulge element are the major determinants for tat activation. RNA gel retardation analysis demonstrates that both tat and cellular proteins bind to TAR RNA, but the mechanism by which these proteins increase HIV gene expression is unknown. We have fractionated HeLa cell nuclear extracts in an attempt to identify cellular proteins that bind to TAR RNA and are involved in regulating HIV gene expression. RNA gel retardation and UV cross-linking reveal that a cellular protein of 185 kD, which we designate TAR RNA-binding protein 185 (TRP-185), binds with both high affinity and marked specificity to TAR RNA. RNA gel retardation and competition analyses indicate that TRP-185 binding is strongly dependent on the TAR RNA loop sequences. The binding of TRP-185 is modulated by both a set of cellular cofactors and the tat protein. Highly purified preparations of TRP-185 are capable of activating in vitro transcription of wild-type, but not mutated, HIV LTR chloramphenicol acetyltransferase (CAT) constructs. These results characterize a positively acting cellular RNA-binding factor, TRP-185, which is involved in the regulation of HIV gene expression.

Amino Acid Sequence↗
PubMed1991

[Constituents of the root of Berneuxia thibetica Decne].

Three crystals have been isolated from the root of Berneuxia thibetica. On the basis of spectral data, crystals A and B have been identified as chrysosplenetin B and 22,23-dihydrospinasterone respectively, while crystal C is a mix of spinasterol and 22,23-dihydrospinasterol.

Drugs, Chinese Herbal↗
PubMed1990

Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kappa B binding motifs.

A primary site of infection by human adenoviruses is lymphoid cells. However, analysis of the viral control elements and the cellular factors that regulate adenoviral gene expression in lymphocytes has not been reported. The adenovirus early region 3 (ES) gene products are involved in the maintenance of viral persistence by complexing with the class I MHC antigens, thus preventing their cell surface expression with a resultant decrease in host immunologic destruction. To determine whether different cellular factors were involved in E3 regulation in lymphocytes as compared with HeLa cells, both DNA binding and transfection analysis with the E3 promoter in both cell types were performed. These studies detected two novel domains referred to as L1 and L2 with a variety of lymphoid but not HeLa extracts. Each of these domains possessed strong homology to motifs previously found to bind the cellular factor NF-kappa B. Transfections of E3 constructs linked to the chloramphenicol acetyltransferase gene revealed that mutagenesis of the distal NF-kappa B motif (L2) had minimal effects on promoter expression in HeLa cells, but resulted in dramatic decreases in expression by lymphoid cells. In contrast, mutagenesis of proximal NF-kappa B motif (L1) had minimal effects on gene expression in both HeLa cells and lymphoid cells but resulted in a small, but reproducible, increase in gene expression in lymphoid cells when coupled to the L2 mutation. Reversing the position and subsequent mutagenesis of the L1 and L2 domains indicated that the primary sequence of these motifs rather than their position in the E3 promoter was critical for regulating gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus Early Proteins↗
PubMed1990

A transdominant tat mutant that inhibits tat-induced gene expression from the human immunodeficiency virus long terminal repeat.

Regulation of human immunodeficiency virus (HIV) gene expression is dependent on specific regulatory regions in the long terminal repeat. These regions include the enhancer, SP1, "TATA," and trans-activating (TAR) regions. In addition, viral regulatory proteins such as tat and rev are important in regulating HIV gene expression. The mechanism of tat activation remains the subject of investigation, but effects at both transcriptional and posttranscriptional levels seem likely. Previous mutagenesis of the tat protein revealed that the amino terminus, the cysteine-rich domain, and the basic domain were all required for complete tat activation. Mutants of other viral trans-acting regulatory proteins, including E1A, tax, and VM65, have been identified that were capable of antagonizing the activity of their corresponding wild-type proteins. We wished to determine whether mutants of the tat protein could be identified that exhibited a similar phenotype. One mutant (delta tat) that truncated the basic domain of tat resulted in a transdominant phenotype inhibiting tat-induced gene expression of the HIV long terminal repeat but not other viral promoters. This mutant exhibited its maximal phenotype in cotransfection experiments when present in an 8- to 30-fold molar excess over the wild-type tat gene. Trans-activation of the HIV long terminal repeat by delta tat was very defective at the DNA concentrations used in these experiments. RNase protection analysis indicated that this mutant decreased tat-induced steady-state mRNA levels of the HIV long terminal repeat. Second-site mutations of the delta tat gene in either the amino terminus or cysteine region eliminated the transdominant phenotype. In contrast to tat, which was localized predominantly to the nucleolus, delta tat was present in both the nucleus and cytoplasm, suggesting that it may inhibit tat function by preventing nucleolar localization. Transdominant mutants of tat may have a role in potentially inhibiting HIV gene expression.

Amino Acid Sequence↗
PubMed1990

Peer interactions and friendships in an ethnically diverse school setting.

Peer interaction, social status, and reciprocated friendships were examined in 2 ethnically diverse groups of children: 104 kindergartners and 106 third graders. There was no correlation between social status and ethnicity. Third-grade children formed more cross-ethnic friendships and engaged in more cross-ethnic positive peer interaction than kindergartners. Children with more cross-ethnic friendships and more cross-ethnic peer interaction were no different in social status than children with fewer cross-ethnic experiences.

Child↗
PubMed1990

[Detection of serum antibodies in jirds against antigens of Brugia malayi].

The indirect fluorescent antibody test (IFA) using the infective larva (L3), adult worm (A) and microfilaria (Mf) antigens was applied to detect anti-filarial antibodies in jirds (Meriones unguiculatus) infected with Brugia malayi. The antibodies against L3 appeared within 3 weeks after infection, followed by A and Mf. The serum antibody levels in the jirds with positive infection were compared with those in the jirds with negative infection. Before appearance of microfilaria, there was insignificant difference between these two groups. However, significant difference was observed after the appearance of microfilaria. The method of sonicating L3 and Mf fragment antigens used in this study is simple, and its results are easy to observe.

Animals↗
PubMed1989

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression.

Base Sequence↗
PubMed1989

Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat.

Five regions of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) have been shown to be important in the transcriptional regulation of HIV in HeLa cells. These include the negative regulatory, enhancer, SP1, TATA, and TAR regions. Previous studies in which purified SP1 was used showed that the three SP1-binding sites in the HIV LTR were important in the in vitro transcription of this promoter. However, no studies to ascertain the role of each of these SP1-binding sites in basal and tat-induced transcriptional activation in vivo have been reported. To determine the role of SP1 sites in transcriptional regulation of the HIV LTR in vivo, these sites were subjected to oligonucleotide mutagenesis both individually and in groups. The constructs were tested by DNase I footprinting with both oligonucleotide affinity column-purified SP1 and partially purified HeLa extract and by chloramphenicol acetyltransferase assays in both the presence and absence of the tat gene. Mutagenesis of each SP1-binding site resulted in minimal changes in basal and tat-induced transcriptional activation. Mutations involving alterations of SP1 sites I and II, I and III, or II and III also resulted in minimal decreases in basal and tat-induced transcriptional activation. However, mutagenesis of all three SP1-binding sites resulted in a marked decrease in tat induction. The latter mutation also greatly decreased DNase I protection over the enhancer, TATA, and TAR regions when partially purified HeLa nuclear extract was used. Mutagenesis of the HIV LTR SP1 sites which converted them to consensus high-affinity SP1-binding sites with the sequence GGGGCGGGGC resulted in increased tat-induced gene expression compared with the wild-type HIV LTR template. These results suggest that SP1, through its interaction with other DNA-binding proteins, is critical for in vivo transcriptional regulation of HIV.

Base Sequence↗
PubMed1988

Alterations in binding characteristics of the human immunodeficiency virus enhancer factor.

Five regions of the human immunodeficiency virus (HIV) long terminal repeat (LTR) serve as binding sites for cellular proteins as demonstrated by DNase I footprinting. These include the negative regulatory, enhancer, SP1, TATA, and untranslated regions. The HIV enhancer region contains two direct repeats of a sequence, GGGACTTTCC, which is also found in the enhancer sequences of simian virus 40, cytomegalovirus, and the immunoglobulin kappa gene. To further characterize binding to the enhancer sequences in the HIV LTR, DNase I footprinting was performed using extracts prepared from several different cell lines. Extracts prepared from lymphoid cells gave altered binding over the enhancer region as compared with extracts prepared from either monocytes or HeLa cells. This altered binding in extracts prepared from lymphoid cells resulted in protection of both direct repeats in the HIV LTR in contrast to complete protection of only one direct repeat with HeLa cell extracts. When HeLa cells were treated with phorbol esters in either the presence or absence of the protein synthesis inhibitor cycloheximide, the binding characteristics over the enhancer element became similar to those seen in extracts prepared from lymphoid cells. These results suggest that phorbol esters may induce posttranslational modifications of cellular transcription factors that alter their DNA-binding characteristics.

Cells, Cultured↗
PubMed1988

Radioimmunodetection of human melanoma with indium-111-labeled monoclonal antibody.

The purpose of the study was threefold: (1) to evaluate the efficacy of an 111In-labeled murine monoclonal antibody (ZME-018) directed against a heavy molecular weight melanoma associated glycoprotein in localizing metastatic disease; (2) to determine the effect of unlabeled antibody mass (2.5, 5, 10, 20, and 40 mg) on labeled antibody blood clearance, biodistribution and lesion detection; (3) to estimate radiation dosimetry. Twenty-five patients with previously documented disease received an intravenous infusion of 2.5 to 40 mg of monoclonal antibody with 1 mg of the antibody labeled with 5 mCi of 111In. There were no acute reactions. Patients were scanned without computer enhancement or background subtraction techniques at 24 and 72 hr after injection. Imaging detected tumor in 14/18 (78%) patients with active disease, identified 24/44 (77%) of lesions greater than 1 cm and changed or specifically directed patient management in 22% (4/18) patients with tumor. There was a prolongation in blood clearance associated with decreased liver and spleen activity following administration of 20 and 40 mg of antibody compared to the three lower antibody dose levels. Assuming a biodistribution similar to [111In]ZME-018, the radiation dose delivered to normal tissues by [90Y]ZME-018 would restrict its use as a routine vehicle for radioimmunotherapy; however, it may be possible to deliver substantial tumor doses in selected patients.

Adult↗
PubMed1987

Upstream regulatory regions required to stabilize binding to the TATA sequence in an adenovirus early promoter.

Of the five early adenovirus promoters, the early region 3 (E3) promoter is one of the most strongly induced by the E1A protein. To identify cellular proteins involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, DNase I footprinting using partially purified Hela cell extracts was performed. Four regions of the E3 promoter serve as binding domains for cellular proteins. These regions are found between -156 to -179 (site IV), -83 to -103 (site III), -47 to -67 (site II), and -16 to -37 (site I), relative to the start of transcription. Examination of the DNA sequences in each binding domain suggests that site III likely serves as a binding site for activator protein 1 (AP-1), site II for the cyclic AMP regulatory element binding protein (CREB), and site I for a TATA binding factor. The factors binding to either site II or III were sufficient to stabilize binding to the TATA sequence (site I). Mutagenesis studies indicated that both sites II and III, in addition to site I, are needed for complete basal and E1A-induced transcription. These results suggest that multiple cellular factors are involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, and that either of two upstream regions are capable of stabilizing factor binding to the TATA sequence.

Adenovirus Early Proteins↗
PubMed1986

Potential pitfall of DMSA scintigraphy in patients with ureteral duplication.

A 5-wk-old male presented with radiographic findings of a duplicated collecting system. A [99mTc]DMSA scan was requested to evaluate cortical function. Images obtained immediately. postinjection showed activity restricted to the upper poles; in contrast, delayed images at 4 hr showed activity in the bladder and throughout both kidneys. Catheterizing the patient drained the activity from the bladder but had little effect on the refluxed renal activity. The early [99mTc]DMSA images were critical in making the proper interpretation. Technetium-99m DMSA is excreted into the urine and this fact needs to be considered when interpreting scans of patients with possible reflux or obstruction. When DMSA scans are obtained in pediatric patients with possible reflux, catheterization prior to the study and early images prior to the appearance of DMSA in the collecting system are recommended.

Humans↗