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Biomedical subjects

S Li

Publications and source records attributed to S Li.

At least 811 records · Page 45Linked to original sources
PubMed1998

Recombinant human respiratory syncytial virus (RSV) from cDNA and construction of subgroup A and B chimeric RSV.

Infectious human respiratory syncytial virus (RSV) was produced from a cDNA clone that contains 15,222 nucleotides of RSV genome derived from the A2 strain of subgroup A. Recovery of infectious RSV from cDNA required cotransfection of only three expression plasmids encoding the nucleoprotein (N), the phosphoprotein (P), and the major polymerase protein (L). Inclusion of the M2-1 plasmid was not required in the transfection reaction and if included did not significantly increase the rescue efficiency. However, a single nucleotide substitution in the RSV leader region (C to G at position 4 in the antigenomic sense), greatly increased the amount of infectious virus recovered from cDNA. A recombinant RSVA2 virus that expresses an additional structural G protein derived from a subgroup B RSV was also obtained. Both A2 and B strain G glycoproteins were expressed in cells infected with the chimeric RSV. A chimeric RSV that expresses a heterologous subgroup antigen in a live attenuated vaccine candidate may be important for prevention of diseases associated with both RSV subgroup A and subgroup B infection.

Animals↗
PubMed1998

Tacrolimus-based partial conditioning produces stable mixed lymphohematopoietic chimerism and tolerance for cardiac allografts.

BACKGROUND: Thoracic organ transplantation remains limited by the reciprocal problems of rejection and the toxicities of nonspecific immunosuppression. Mixed bone marrow chimerism reliably produces donor-specific transplantation tolerance without immunosuppressive drugs. We have previously described a nonmyeloablative conditioning regimen based on recipient treatment with antilymphocyte serum, tacrolimus, and low-dose total-body irradiation that yields long-term multilineage allogeneic bone marrow chimerism in the rat. We have now investigated whether mixed bone marrow chimerism that arises from this partial conditioning strategy produces permanent acceptance of donor-specific cardiac allografts. METHODS AND RESULTS: Mixed allogeneic chimeras (ACI-->WF) were prepared by treating Wistar Furth recipients with a single dose of antilymphocyte serum 5 days before bone marrow transplantation and tacrolimus 1 mg/kg/d from days -1 to 10. Five hundred cGy total-body irradiation was administered immediately before infusion of 1 x 10(8) donor (ACI) T-cell depleted marrow cells. All recipients were chimeric, with a mean level of donor chimerism = 26.3 +/- 3.5%. Chimeras underwent heterotopic cardiac transplantation 4 weeks after bone marrow transplantation. All donor-specific (ACI) grafts were permanently accepted (follow-up, 230 to 360 days). Third-party grafts were rapidly rejected. Histology of long-surviving donor-specific grafts was without evidence of acute or chronic rejection. Second-set donor-specific skin grafts transplanted to chimeras 135 days after heart transplantation showed long-term survival (> 130 days), whereas third-party skin grafts were rapidly rejected. Mixed lymphocyte reaction demonstrated in vitro donor-specific hyporeactivity. CONCLUSIONS: A tacrolimus-based nonmyeloablative recipient conditioning regimen produces mixed bone marrow chimerism and donor-specific tolerance to cardiac allografts in the rat.

Animals↗
PubMed1998

Kenny-Caffey syndrome and microorchidism.

We report on two adolescent boys with Kenny-Caffey syndrome and microorchidism. The first patient had elevated levels of serum follicle-stimulating hormone, but normal levels of luteinizing hormone and testosterone. There was no evidence of a microdeletion of the Y chromosome. The second patient had Leydig cell hyperplasia with normal seminiferous tubules and spermatogenesis, and normal pituitary histologic findings at autopsy. The presence of microorchidism in these patients confirms the previous observations and suggests subfertility, but does not fully clarify the pathogenesis.

Abnormalities, Multiple↗
PubMed1998

Adenoviral-mediated gene transfer in lymphocytes.

Although adenovirus can infect a wide range of cell types, lymphocytes are not generally susceptible to adenovirus infection, in part because of the absence of the expression of the cellular receptor for the adenoviral fiber protein. The cellular receptor for adenovirus and coxsackievirus (CAR) recently was cloned and shown to mediate adenoviral entry by interaction with the viral fiber protein. We show that the ectopic expression of CAR in various lymphocyte cell lines, which are almost completely resistant to adenovirus infection, is sufficient to facilitate the efficient transduction of these cells by recombinant adenoviruses. Furthermore, this property of CAR does not require its cytoplasmic domain, consistent with the idea that CAR primarily serves as a high affinity binding site for the adenoviral fiber protein, and that viral entry is mediated by interaction of the viral penton base proteins with cellular integrins. As a demonstration of their functional utility, we used CAR-expressing lymphocytes transduced with an adenovirus expressing Fas ligand to efficiently kill Fas receptor-expressing tumor cells. The ability to efficiently manipulate gene expression in lymphocyte cells by using adenovirus vectors should facilitate the functional characterization of pathways affecting lymphocyte physiology.

Adenoviridae↗
PubMed1998

Activation of the insulin-like growth factor type 1 receptor by deletion of amino acids 870-905.

We have created a deletion mutant of the insulin-like growth factor type 1 receptor (IGF-1 R) which lacks the 36 amino acids (aa) immediately N-terminal to the transmembrane domain (Delta870-905 IGF-1 R). This region has been reported to have a negative effect on the transforming potential of an avian sarcoma virus gag-IGF-1 R fusion protein. We have sought to determine whether this region plays a similar role in the intact IGF-1 R. Analysis of the tyrosine kinase activity of the Delta870-905 IGF-1 R shows that the mutant receptor is autophosphorylated without IGF-1 stimulation, indicating that the tyrosine kinase domain is constitutively active. In addition, processing of the receptor is decreased, resulting in accumulation of a high molecular weight proreceptor containing both alpha and beta-subunits. A well-characterized substrate of the IGF-1 R, IRS-1, is constitutively phosphorylated by the Delta870-905 IGF-1 R and phosphoinositide (PI) 3-kinase activity, which is normally activated by the phosphorylation of IRS-1 following IGF-1 stimulation, is increased even in the absence of IGF-1. A second intracellular signal pathway normally activated by IGF-1, the MAP kinase pathway, showed no increase in activity in the absence of IGF-1. The Delta870-905 IGF-1 R promoted cell proliferation only in the presence of IGF-1. We conclude that this deletion increases the basal activity of the IGF-1 receptor tyrosine kinase and activates PI 3-kinase, but is unable to stimulate MAP kinase in the absence of ligand. These results confirm those seen in the gag-IGF-1 R fusion protein and indicate that aa 870-905 exert a negative effect on the tyrosine kinase domain of the beta-subunit of the IGF-1 R.

Amino Acid Sequence↗
PubMed1998

Mutational analysis of caveolin-induced vesicle formation. Expression of caveolin-1 recruits caveolin-2 to caveolae membranes.

Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50-100 nm. Although recombinant expression of caveolin-1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin-caveolin interactions regulate the size of caveolae, as caveolin-1 undergoes two stages of self-oligomerization. To test this hypothesis directly, we have created two caveolin-1 deletion mutants that lack regions of caveolin-1 that are involved in directing the self-assembly of caveolin-1 oligomers. More specifically, Cav-1 delta61-100 lacks a region of the N-terminal domain that directs the formation of high molecular mass caveolin-1 homo-oligomers, while Cav-1 deltaC lacks a complete C-terminal domain that is required to allow caveolin homo-oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav-1 delta61-100 and Cav-1 deltaC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500-1000 nm. In addition, caveolin-induced vesicle formation appears to be isoform-specific. Recombinant expression of caveolin-2 under the same conditions failed to drive the formation of vesicles, while caveolin-3 expression yielded caveolae-sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin-1 expression, but continue to express caveolin-2, no morphologically distinguishable caveolae are observed. In addition, as caveolin-2 alone exists mainly as a monomer or homo-dimer, while caveolins 1 and 3 exist as high molecular mass homo-oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae-sized vesicles. In direct support of this notion, regulated induction of caveolin-1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin-2 to caveolae membranes. The ability of caveolin-1 to recruit caveolin-2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co-expressed and interact with each other to form high molecular mass hetero-oligomers containing both caveolins 1 and 2.

3T3 Cells↗
PubMed1998

A unifying Tm diagram for phosphatidylethanolamines with sn-1 C20 saturated and sn-2 C18 unsaturated acyl chains.

We have determined calorimetrically the phase transition temperature (Tm) values of five sn-1 saturated/sn-2 unsaturated phosphatidylethanolamines (PE) in which the sn-1 acyl chain has 20 carbons and the sn-2 acyl chain has 18 carbons with different number and position of the cis double bond. When these Tm values are combined with the five published Tm values of related unsaturated PE, a unifying Tm diagram is generated for the first time. Moreover, as the molecular mechanics simulated structures of these lipids are taken into consideration, this unifying Tm diagram provides insight into how variations in the number and position of the cis double bond in the lipid's sn-2 acyl chain can influence the phase transition behavior of the lipid bilayer.

Calorimetry, Differential Scanning↗
PubMed1998

Major histocompatibility complex class II expression by intrinsic renal cells is required for crescentic glomerulonephritis.

The requirement for major histocompatibility complex class II (MHC II) to initiate immune renal injury was studied in a murine model of CD4(+) T cell-dependent crescentic glomerulonephritis (GN). C57BL/6 (MHC II+/+) mice developed crescentic GN with glomerular CD4(+) T cell infiltration and renal injury, in response to a nephritogenic antigen (sheep globulin) planted on their glomerular basement membrane. MHC II-deficient C57BL/6 mice (MHC II-/-) did not develop crescentic GN, CD4(+) T cell infiltration, or injury, indicating that this form of immune glomerular injury is MHC II dependent. The requirement for MHC II expression by intrinsic renal cells was studied in chimeric mice, which expressed MHC II on bone marrow-derived cells and in the thymus, but not in the kidneys. These chimeric mice had normal T and B cell populations and MHC II expression in their spleens and lymph nodes and developed an immune response to systemically and cutaneously administered sheep globulin. However, they did not develop crescentic GN, CD4(+) T cell infiltration, or renal injury in response to the sheep globulin planted in their glomeruli. These studies demonstrate that interaction of CD4(+) T cells with intrinsic renal cells expressing MHC II is required for development of cell-mediated immune renal injury.

Animals↗
PubMed1998

Calorimetric studies of phosphatidylethanolamines with saturated sn-1 and dienoic sn-2 Acyl chains.

We have semi-synthesized 18 species of mixed chain phosphatidylethanolamines (PE) in which the sn-1 acyl chain is derived from stearic, arachidic, and behenic acids, and the sn-2 acyl chain is originated from cis,cis-octadecadienoic and cis, cis-eicosadienoic acids with the two methylene-interrupted double bonds located at various positions. These PEs constituting the bilayers in the aqueous dispersion were subjected to differential scanning calorimetric experiments. The Tm values associated with the gel-to-liquid crystalline phase transitions for these PEs are found to be significantly smaller than those of the saturated counterparts. Moreover, the magnitude of the Tm-lowering effect of acyl chain diunsaturation depends critically on the positions of the two methylene-interrupted cis double bonds in the sn-2 acyl chain. Specifically, if the sn-2 acyl chain is derived from cis, cis-octadecadienoic acid, the Tm-lowering effect has the following decreasing order: Delta9,12 > Delta6,9 > Delta12,15. For cis, cis-eicosadienoyl acyl chain, the Tm-lowering effect is stronger in the order of Delta10,13 > Delta11,14 > Delta8,11 > Delta5,8 > Delta14,17. Finally, a refined molecular model is presented that can adequately explain the Tm-lowering effect of sn-2 acyl chain diunsaturation. Moreover, this same refined molecular model can also be invoked to better interpret the Tm-lowering effect observed for sn-1 saturated/sn-2 monoenoic PE.

Calorimetry, Differential Scanning↗
PubMed1998

Theonellapeptolide IIIe, a new cyclic peptolide from the New Zealand deep water sponge, Lamellomorpha strongylata.

The structure, stereochemistry, and conformation of theonellapeptolide IIIe (1), a new 36-membered ring cyclic peptolide from the New Zealand deep-water sponge Lamellomorpha strongylata, is described. The sequence of the cytotoxic peptolide was determined through a combination of NMR and MS-MS techniques and confirmed by X-ray crystal structure analysis, which, with chiral HPLC, established the absolute stereochemistry.

Amino Acids↗
PubMed1998

Identification of the CD8 DE loop as a surface functional epitope. Implications for major histocompatibility complex class I binding and CD8 inhibitor design.

We used an approach of protein surface epitope mapping by synthetic peptides to analyze the surface structure-function relationship of the CD8 protein. Small synthetic peptide mimics of the CD8 DE loop were shown to effectively block CD8 binding to major histocompatibility complex (MHC) class I molecules and possess significant inhibitory activity on in vitro CD8(+) T cell function. These results suggested that the DE loop region of the CD8 protein is an important functional epitope mediating CD8-MHC class I interaction and the activation of CD8(+) T cells, a finding that is consistent with the recently reported crystal structure of the CD8-MHC class I complex. The structural basis for the biological activity of the DE loop peptide was further analyzed in a series of analogs containing alanine substitutions. This study provides support for the concept of bioactive peptide design based on protein surface epitopes and suggests that such an approach may be applicable to other protein-protein complexes, particularly those of immunoglobulin superfamily molecules.

Amino Acid Sequence↗
PubMed1998

Collagen/apatite coating on 3-dimensional carbon/carbon composite.

A three-dimensional carbon/carbon composite (3D C/C) was studied as potential bone-repairing material; its major mechanical properties were found to be closer to those of human bone than other common bone-repairing materials available. In vitro calcification tests revealed that as-received 3D C/C is almost bioinert in simulated body fluid (SBF) over an immersion period of 4 weeks. To improve the bioactivity of 3D C/C, surface modification was accomplished through two practical routes: (1) grafting with polyethylene glycol (PEG) and (2) phosphorylation and precalcification. After grafting with alpha, omega di(aminopropyl) polyethylene glycol 800 (NH2-PEG-NH2), a continuous layer of calcium phosphate was formed on the surface of 3D C/C in SBF after 4 weeks. Phosphorylated 3D C/C samples have the ability to induce apatite precipitation after precalcification in a saturated Ca(OH)2 solution for 1 week. To speed up the coating process, a calcification solution with collagen was developed in which a collagen/apatite coating layer can be formed on 3D C/C in 9 h in ambient conditions.

Apatites↗
PubMed1998

Role of nitric oxide in the regulation of gonadotropin-releasing hormone and tyrosine hydroxylase gene expression in the male rat brain.

It has been recently demonstrated that nitric oxide (NO), a free radical gas which may act as neurotransmitter in the brain, can stimulate the in vivo release of luteinizing hormone as well as the in vitro hypothalamic release of gonadotropin-releasing hormone (GnRH). In order to study the influence of NO on GnRH mRNA expression, two inhibitors of NO synthase (NOS) NG-monomethyl-l-arginine (NMMA) and HP-228, were microinjected into the left lateral ventricle of sham-operated and castrated male rats 4 h before sacrifice. Since the dopaminergic system can positively influence GnRH gene expression, we have also measured in the same animals tyrosine hydroxylase (TH) mRNA in tuberoinfundibular dopamine (TIDA) neurons. GnRH and TH mRNA levels were measured at the cellular level by quantitative in situ hybridization. The injection of HP-228 or NMMA induced a similar decrease (-19.5%) in GnRH mRNA. In castrated animals, the hybridization signal was 88% higher than that observed in sham-operated animals. Both HP-228 and NMMA produced in castrated animals a 39% decrease in GnRH mRNA. In contrast the injection of NOS inhibitors resulted in an increase in the amount of TH mRNA in TIDA neurons. The stimulating effect was more striking in HP-228-treated (+60%) than in NMMA-treated (+32%) animals. Castration did not induce any changes in the number of silver grains overlying TIDA neurons, while the administration of either HP-228 or NMMA induced a 43% increase in castrated animals. These results together with previous ones on GnRH release in vitro suggest that NO exerts a positive influence not only on the secretion but also on the biosynthesis of GnRH. Since NO appears to play a role in the negative regulation of dopamine, it is likely that the increase in GnRH mRNA expression is not mediated by the TIDA system.

Animals↗
PubMed1998

Specific detection of phosphatidylinositol 3,4,5-trisphosphate binding proteins by the PIP3 analogue beads: an application for rapid purification of the PIP3 binding proteins.

Phosphatidylinositol (PI) 3-kinase is known as one of the key molecules involved in the various biological events such as vesicle trafficking, cytoskeletal rearrangements and cell survival. T clarify the molecular basis underlying these events, we have tried to identify the proteins that can interact with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the lipid product of PI3-kinase. Using a new PIP3 analogue, PIP3-APB, we synthesized an affinity column for PIP3 binding proteins. This enabled us to purify and identify several PIP3 binding proteins such as Tec tyrosine kinase, Gap1m, and Akt, as the candidates for the downstream molecules of PI3-kinase. All of these proteins contain PH domains, possible binding sites for phospholipids. Studies with various deletion mutants of Tec or Gap1m revealed that their PH domains are indeed the binding sites for PIP3. These results demonstrate that this PIP3-analogue binds various PIP3 binding proteins with high specificity and may be useful to elucidate the downstream mechanisms of PI3-kinases-mediated signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1998

[Association between angiotensin II type I receptor gene and human essential hypertension].

OBJECTIVE: To determine whether the angiotensin II type I receptor gene might be implicated in human essential hypertension. METHODS: This was a case-control study on hypertension and normal blood pressure in Chinese DNA abstracted samples from 51 cases of essential hypertension and 74 normal controls were analysed by polymerase chain reaction,digestion of restriction enzyme and electrophesis. RESULTS: The genotype frequency of 1166A/1166C was 0. 196 (10/51) in the essential hypertension group, 0.081 (6/74) in the control group. The genotype frequency of 1166C/1166C was 0 in both groups. The gene frequency of 1166C was 0.098 in the essential hypertension group, 0.041 in the controls. There was a significant increase in allelic frequency of 1166C in hypertension. CONCLUSION: The results indicate that the increase in allelic frequency of 1166C is a risk factor and hence suggest that the change in the angiotensin II type I receptor gene is associated with essential hypertension.

Adolescent↗
PubMed1998

Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly.

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a beta-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their amino-termini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti- and oncoviruses mature via analogous pathways.

Amino Acid Sequence↗
PubMed1998

Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs.

Active transport of proteins into the nucleus is mediated by interaction between the classical nuclear localization signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex. This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival. Using a fragment of BRCA1 containing the two NLS motifs as a bait for yeast two-hybrid screening, we have isolated four clones, one of which is importin alpha. Here we characterize one of the other clones identified, BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in human mammary epithelial cells and some but not all tissues of mice. The isolated full-length cDNA encodes a novel protein containing 600 amino acid residues with pI 6.04. Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats are present in the middle and C-terminal regions of the protein, respectively. BRAP2 also shares significant homology with a hypothetical protein from yeast Saccharomyces cerevisiae, especially in the zinc finger region. Antibodies prepared against the C-terminal region of BRAP2 fused to glutathione S-transferase specifically recognize a cellular protein with a molecular size of 68 kDa, consistent with the size of the in vitro translated protein. Cellular BRAP2 is mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar specificity to that of importin alpha in both two-hybrid assays in yeast and glutathione S-transferase pull-down assays in vitro. Other motifs such as the SV40 large T antigen NLS motif and the bipartite NLS motif found in mitosin are also recognized by BRAP2. Similarly, the yeast homolog of BRAP2 also binds to these NLS motifs in vitro. These results imply that BRAP2 may function as a cytoplasmic retention protein and play a role in regulating transport of nuclear proteins.

Amino Acid Sequence↗