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S Li

Publications and source records attributed to S Li.

At least 1,009 records · Page 56Linked to original sources
PubMed1997

T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like mechanism.

Premature termination codons (PTCs) are known to decrease mRNA levels. Here, we report our investigation of the mechanism for this downregulation using the TCR-beta gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic development. We found that a mini-gene version of this gene, which contains only three TCR-beta exons, exhibited efficient downregulation in response to PTCs. This demonstrates that the full coding sequence is not necessary for appropriate regulation. Mutation of the translation start AUG and a downstream in-frame AUG that displayed similarity to the Kozak consensus sequence reversed the downregulatory response to PTCs. Thus, an AUG start codon is required to define the reading frame of a PTC. Specific suppressor tRNAs also reversed the downregulatory response, strongly implicating the involvement of a translation-like process. Remarkably, the addition of suppressor tRNAs or the inactivation of the start AUGs caused a dramatic rise in the levels of PTC-bearing transcripts in the nuclear fraction prepared by two independent methods. Collectively, our results provide evidence for a codon-based surveillance mechanism associated with the nucleus that downregulates aberrant transcripts encoding potentially toxic polypeptides from nonproductively rearranged genes.

Animals↗
PubMed1997

Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways.

The interferon-inducible double-stranded RNA protein kinase PKR controls protein synthesis through the phosphorylation of eukaryotic translation initiation factor (eIF)-2. In addition to its demonstrated role in translational control, several reports have suggested a transcriptional role for PKR. Here we report that PKR is involved in IFN- and dsRNA-signaling pathways by modulating the function of the signal transducer and activator of transcription STAT1. We also show that PKR associates with STAT1 in mouse and human cells. The association is not a kinase-substrate interaction since STAT1 phosphorylation is not modified by PKR in vitro or in vivo. In addition, the formation of the PKR-STAT1 complex is not dependent upon the enzymatic activity of PKR but does require the dsRNA-binding domain of PKR. Moreover, there is a concomitant decrease in PKR-STAT1 interaction and increase in STAT1 DNA binding in response to IFNs or dsRNA. These findings suggest that PKR plays an important role in IFN and dsRNA-signaling pathways by modulating the transcriptional function of STAT1.

3T3 Cells↗
PubMed1997

Detection of myocardial perfusion in multiple echocardiographic windows with one intravenous injection of microbubbles using transient response second harmonic imaging.

OBJECTIVES: The purpose of this study was to prove that transient response harmonic imaging could detect normal and abnormal myocardial perfusion in multiple echocardiographic windows with one intravenous injection of microbubbles in humans. BACKGROUND: Myocardial ultrasound contrast can be produced from intravenous perfluorocarbon-exposed sonicated dextrose albumin, and ultrasound can be significantly improved by briefly suspending the interval between frame rates. Whether this contrast can noninvasively quantify myocardial perfusion in humans is unknown. METHODS: In 28 patients, harmonic transient response imaging was used to image the heart in multiple different imaging planes after one intravenous injection of ultrasound contrast agent. Twenty-five of these 28 patients had a repeat injection during dipyridamole stress. In the primary view, the ultrasound transmission rate was one frame per cardiac cycle; in secondary and tertiary views, the transmission rate was once every multiple cardiac cycles. Regional myocardial contrast was visually assessed and quantified off-line. Quantitative rest thallium and dipyridamole stress sestamibi imaging was also performed. RESULTS: Perfusion abnormalities were evident in the secondary and tertiary views only with one frame every multiple cardiac cycles. Regional peak myocardial videointensity (PMVI) correlated closely with regional tracer uptake in individual patients both at rest (r = 0.84) and during stress (r = 0.88). A PMVI ratio (abnormal region divided by the region with highest nuclear uptake) < 0.6 in any view had a 92% sensitivity and a 84% specificity in identifying a regional nuclear perfusion abnormality. CONCLUSIONS: Transient response imaging produces myocardial contrast in multiple views with one intravenous injection of contrast agent and can accurately identify regional myocardial perfusion abnormalities.

Adult↗
PubMed1997

Regulation of proopiomelanocortin gene expression by endogenous ligands of the GABAA receptor complex as evaluated by in situ hybridization in the rat pars intermedia.

The neurotransmitter gamma-aminobutyric acid (GABA) exerts a tonic inhibitory influence on proopiomelanocortin (POMC) neurons in the hypothalamus as well as on the melanotrope cells of the intermediate lobe (IL) of the pituitary gland. Moreover, the activation of the GABAA receptor complex by different ligands has been shown to exert a negative influence on the POMC gene expression at the hypothalamic level. In order to elucidate the in vivo regulation of the POMC mRNA levels in the intermediate lobe of the pituitary by endogenous ligands of the GABAA receptor complex, we have studied the effect of intravenous (i.v.) and intracerebroventricular (i.c.v) injections of octadecaneuropeptide (ODN), a peptide derived from diazepam-binding inhibitor (DBI). The possible involvement of neurosteroids in the action of ODN on melanotropic cells was evaluated following inhibition of two enzymes involved in the biosynthesis of neurosteroids known as activators of G3BAA receptor complex: trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and MK-906, an inhibitor of 5 alpha-reductase. The i.v. injection of ODN produced a dose-dependent inhibition of POMC gene expression in the IL. The i.c.v. injection of ODN also depressed POMC mRNA. These effects were completely reversed by the concomitant administration of the GABAA antagonist picrotoxin. Similar results were obtained in POMC neurons in the arcuate nucleus (AN) of the hypothalamus. Trilostane administration induced an increase in POMC mRNA and also prevented the inhibitory influence of ODN. The neurosteroid pregnenolone-sulfate, a negative modulator of the GABAA receptor, also stimulated POMC gene expression. On the other hand, MK-906 produced a decrease in mRNA levels and could not reverse the effect of ODN. The results indicate that activation of the GABAA receptor complex by the endogenous benzodiazepine receptor ligand ODN can induce a negative regulation of POMC gene expression in the IL of the pituitary and neurons in the AN. The present results do not provide clear evidence that neurosteroids are involved in the action of ODN on POMC gene expression in the IL.

Adrenalectomy↗
PubMed1997

Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins.

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein alpha subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolin-scaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules. Here, we have used the caveolin-scaffolding domain as a receptor to select random peptide ligands from phage display libraries. These caveolin-selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases. A known caveolin-interacting protein, Gi2alpha, was used as a ligand to further investigate the nature of this interaction. Gi2alpha and other G-protein alpha subunits contain a single region that generally resembles the sequences derived from phage display. We show that this short peptide sequence derived from Gi2alpha interacts directly with the caveolin-scaffolding domain and competitively inhibits the interaction of the caveolin-scaffolding domain with the appropriate region of Gi2alpha. This interaction is strictly dependent on the presence of aromatic residues within the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolin-scaffolding domain. In addition, we have used this interaction to define which residues within the caveolin-scaffolding domain are critical for recognizing these peptide and protein ligands. Also, we find that the scaffolding domains of caveolins 1 and 3 both recognize the same peptide ligands, whereas the corresponding domain within caveolin-2 fails to recognize these ligands under the same conditions. These results serve to further demonstrate the specificity of this interaction. The implications of our current findings are discussed regarding other caveolin- and caveolae-associated proteins.

Amino Acid Sequence↗
PubMed1997

High-efficiency stable DNA transfection using cationic detergent and glycerol.

We show that a brief exposure to a simple cationic detergent, in combination with a glycerol 'shock', can result in high frequencies of stable DNA transfection into mammalian cells. Using both Chinese hamster and human cell lines, frequencies of 1 per 1000, or even 1 per 100, viable cells can be achieved readily after optimizing the transfection conditions in a simple buffer. The mechanism of DNA transfer is proposed to differ from that for polycationic liposomes: as well as supplying a hydrophobic counterion, CDAB may promote a conformational change in DNA, allowing the DNA to be readily taken up after the glycerol shock to the cell.

Animals↗
PubMed1997

Mutational analysis of the properties of caveolin-1. A novel role for the C-terminal domain in mediating homo-typic caveolin-caveolin interactions.

Caveolin is a principal structural component of caveolae membranes in vivo. Recently, a family of caveolin-related proteins has been identified; caveolin has been retermed caveolin-1. Caveolin family members share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes. Here, we have used a deletion mutagenesis approach as a first step toward understanding which regions of caveolin-1 contribute to its unusual properties. Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1DeltaC) or the N-terminal domain (Cav-1DeltaN); these mutants were compared with the behavior of full-length caveolin-1 (Cav-1FL) expressed in parallel. Our results show that the N-terminal domain and membrane spanning segment are sufficient to form high molecular mass oligomers of caveolin-1. However, a complete caveolin-1 molecule is required for conveying detergent insolubility and incorporation into low density Triton-insoluble complexes. These data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an unknown role for the C-terminal domain in this process. To better understand the role of the C-terminal domain, this region of caveolin-1 (residues 135-178) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Purified recombinant glutathione S-transferase-C-Cav-1 was found to stably interact with full-length caveolin-1 but not with the two caveolin-1 deletion mutants. These results suggest that the C-terminal domain interacts with both the N-terminal and C-terminal domains of an adjacent caveolin-1 homo-oligomer. This appears to be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3. Homo-typic interaction of the C-terminal domain with an adjacent homo-oligomer could provide a mechanism for clustering caveolin-1 homo-oligomers while excluding other caveolin family members. This type of lateral segregation event could promote caveolae membrane formation and contribute to the detergent insolubility of caveolins-1, -2, and -3.

Animals↗
PubMed1997

Hydrophobic chromatography of the HL-60 cellular fraction co-binding with hexamethylene bisacetamide.

Methods of separating N-acetyl-1,6-diaminohexane (NADAH) and its immobilization to diol-silica have been developed. Hexamethylene bisacetamide (HMBA) and its metabolite NADAH are used as inducers of leukemia cell differentiation. The inducing mechanism of HMBA is still not clear. Experiments show that HMBA and NADAH undergo relatively strong hydrophobic reactions and do not readily undergo ion-exchange with the proteins of the cytosolic fraction of HL-60 cells during immobilization of NADAH; the retention time of the proteins was longer than that of the phosphatides. These results show that the adsorption of HMBA and NADAH to proteins was higher than that to phosphatides. The expected biospecific receptor binding with HMBA has not been found.

Acetamides↗
PubMed1997

A computer screening approach to immunoglobulin superfamily structures and interactions: discovery of small non-peptidic CD4 inhibitors as novel immunotherapeutics.

The interaction between CD4 and major histocompatibility complex (MHC) class II proteins is critical for the activation of CD4+ T cells, which are involved in transplantation reactions and a number of autoimmune diseases. In this study we have identified a CD4 surface pocket as a functional epitope implicated in CD4-MHC class II interaction and T-cell activation. A computer-based strategy has been used to screen approximately 150,000 non-peptidic organic compounds in a molecular data base and to identify a group of compounds as ligands of the proposed CD4 surface pocket. These small organic compounds have been shown to specifically block stable CD4-MHC class II binding, and exhibit significant inhibition of immune responses in animal models of autoimmune disease and allograft transplant rejection, suggesting their potential as novel immunosuppressants. This structure-based computer screening approach may have general implications for studying many immunoglobulin-like structures and interactions that share similar structural features. Furthermore, the results from this study have demonstrated that the rational design of small non-peptidic inhibitors of large protein-protein interfaces may indeed be an achievable goal.

Animals↗
PubMed1997

A yeast artificial chromosome (YAC) contig encompassing the critical region of the X-linked lymphoproliferative disease (XLP) locus.

X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein-Barr virus (EBV) infection. Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma. Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence. From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated. Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients. Three more families with interstitial deletions were found. Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescence in situ hybridization and which consists of 15 overlapping YAC clones, has been constructed. A detailed restriction enzyme map of the region has been constructed. YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction enzymes. The STSs and lambda subclones were used in Southern hybridization and PCR analyses. The work presented here substantially refines the critical region for XLP. The YAC contig with the overlapping interstitial deletions constitutes the basis for the construction of a transcriptional map of the critical region and facilitates the identification of the XLP gene.

Chromosomes, Artificial, Yeast↗
PubMed1997

The state of maturation of monocytes into macrophages determines the effects of IL-4 and IL-13 on HIV replication.

The molecular mechanisms of the effects of IL-4 and IL-13 on HIV infection in human monocytes as they matured into monocyte-derived macrophages over 7 days were investigated using HIV-1(BaL), and low passage clinical strains. IL-4 and IL-13 up-regulated the expression of both genomic and spliced HIV mRNA in monocytes cultured on Teflon, as determined by Northern analysis and p24 Ag assay. Using a nuclear run-on assay, IL-4 stimulation was shown to enhance transcription by two- to threefold. IL-4 stimulated nuclear factor-kappaB nuclear translocation and binding before enhancement of HIV RNA expression. Conversely, IL-4 and IL-13 markedly and significantly inhibited HIV replication at the transcriptional level in monocyte-derived macrophages, and this occurred whether these cytokines were added before or after HIV infection. The reversal from stimulation to inhibition occurred after 3 to 5 days of adherence to plastic. IL-4 had no significant effect on HIV reverse transcription. The effect of both cytokines on the monocyte maturation/differentiation (CD11b, CD13, and CD26) and other macrophage markers (CD14 and CD68) was examined. IL-4 enhanced CD11b, but inhibited CD26 expression and delayed CD13 loss. IL-13 had similar effects on CD11b and CD13, but no effect on CD26. Hence, these cytokines do not simply enhance monocyte differentiation, but have complex and slightly divergent effects that impact on HIV replication probably through cell signaling pathways and nuclear factor-kappaB translocation.

Cell Differentiation↗
PubMed1997

Immunoglobulin superfamily proteins: structure, mechanisms, and drug discovery.

We review the recent progress made in our laboratories in structure-based drug design targeting proteins of the immunoglobulin superfamily (IgSF). We will focus on the CD4 protein, which is involved in T cell function, as a specific example of how the general concept and methodologies can be applied. Recent studies of CD4 structure and function have revealed new insight into possible mechanisms for CD4 self-association and its role in binding to major histocompatibility complex (MHC) class II molecules and initiation of T cell activation. This has led to the formulation of a hypothetical model of co-oligomerization of CD4, MHC class II, and T cell receptor (TCR). Such a basic understanding of CD4 structure and mechanisms has aided the development of a new generation of potential immunotherapeutics targeting specific CD4 surface functional sites. The design and discovery of small molecular inhibitors of CD4 and other IgSF proteins, in peptide, peptidomimetic, and nonpeptidic organic forms have opened new avenues for chemical research in which peptide, organic, and more recently combinatorial chemistry techniques can be used to further develop these promising lead analogs into a new generation of effective pharmaceuticals.

Animals↗
PubMed1997

HIV infection of macrophages and pathogenesis of AIDS dementia complex: interaction of the host cell and viral genotype.

AIDS dementia complex (ADC) develops in only a third of HIV-infected patients who progress to AIDS. Macrophages and microglial cells are the major cellular sites of productive HIV replication in brain. Using 11 blood isolates of HIV from asymptomatic patients there was marked variation in tropism and the level of productive infection in recently adherent monocytes and monocyte-derived macrophages cultured in vitro. However, less variation was seen with 19 blood isolates from advanced HIV infection and 11 postmortem tissue isolates from brain, cerebrospinal fluid, spleen, and lung. Newly adherent monocytes expressed CCR5 in all seven patients tested, consistent with their susceptibility to infection but not explaining the above variability. There is, also marked regional variability in neuropathology in the brain of patients with ADC. We have demonstrated that there was marked variation in the V3 sequences of HIV clones from different regions of the cortex of a patient with ADC, suggesting independent evolution of HIV replication in brain. Furthermore, production of the neurotoxin quinolinic acid from HIV-infected macrophages varied, depending on the host and source of HIV isolate. Hence variations in viral genotype, production by infected macrophages, and subsequent toxin production may contribute to the variability in neuropathology between individuals and between different regions of the brain in the same individual.

AIDS Dementia Complex↗
PubMed1997

Multi-gradient echo with susceptibility inhomogeneity compensation (MGESIC): demonstration of fMRI in the olfactory cortex at 3.0 T.

Short image acquisition times and sensitivity to magnetic susceptibility favor the use of gradient echo imaging methods in functional MRI (fMRI). However, magnetic susceptibility effects attributed to air-tissue interfaces also lead to severe signal loss in images of the large inferior frontal and lateral temporal cortices of the human brain, which renders these regions inaccessible to fMRI. The signal loss is caused by the local field gradients in the silce selection direction. A multigradient echo with magnetic susceptibility inhomogeneity compensation method (MGESIC) is proposed to overcome this problem. The MGESIC method effectively corrects the susceptibility artifacts and maintains the advantages of gradient echo methods to both BOLD sensitivity and fast image acquisition. The effectiveness of the MGESIC method is demonstrated by fMRI experimental results within the olfactory cortex.

Humans↗
PubMed1997

A method to create an optimum current distribution and homogeneous B1 field for elliptical birdcage coils.

For a birdcage coil with elliptical cross-section, a sinusoidal current pattern does not provide a homogeneous B1 field. A simple theory was developed to create an optimized current distribution for elliptical birdcage coils. This optimized current pattern can create a perfectly homogeneous B1 field inside any elliptical shape. To verify the theory, a 16-element high-pass elliptical birdcage coil was built inside a circular RF shield. The current was optimized by using the inductance characteristics of the coil components to calculate the end-ring capacitances. The B1 field was theoretically calculated and experimentally mapped for the optimized elliptical bird-cage coil and a nonoptimized coil. The results demonstrate that by optimizing the current distribution, a very homogeneous B1 field is produced. This method can be directly applied in design and construction of elliptical birdcage coils for imaging of the naturally occurring elliptical cross-sectional geometries in the human body.

Computer Simulation↗
PubMed1997

In vivo sodium multiple quantum spectroscopy of human articular cartilage.

The authors report, for the first time, sodium properties of human articular cartilage in vivo using sodium multiple-quantum-filtered methods with a surface coil. A flip angle-independent, phase-cycled pulse sequence was used to obtain triple-quantum-filtered spectra as a function of preparation time. Biexponential relaxation rates were calculated by fitting the triple-quantum-filtered spectral amplitudes to a theoretical expression. Theoretical analysis of the flip angle dependence of even rank two-quantum coherence (T2[2]), odd rank two-quantum coherence (T2[3]), and triple-quantum coherence are presented and verified against experimental results on a cartilage specimen. Sodium multiple-quantum-filtered spectral lineshapes obtained in vivo correlate well with those observed on in vitro specimens. Relaxation rates obtained from asymptomatic volunteers were found to be: T(2rise) = 1.0 + 0.12 ms, T(2decay) = 12.0 +/- 0.75 ms (mean +/- SD). The diagnostic potential of this method in detecting early changes in articular cartilage is described.

Animals↗