Biomedical subjects

S Li

Publications and source records attributed to S Li.

At least 235 records · Page 13Linked to original sources

PubMed2001

Minimization of MC1R selectivity by modification of the core structure of alpha-MSH-ND.

BACKGROUND: Melanocortin, through its distinct receptor subtypes, has many different effects. Receptor-selective ligands are required to reduce the undesirable effects of melanocortin. To investigate which conformation is preferable to a given melanocortin receptor subtype, a structural and functional analysis of the ligand-receptor interactions was made by studying the biological activity, the nuclear magnetic resonance structures, and the patterns of the ligand-receptor interaction for each receptor subtype by homology modeling analysis. RESULTS: Among the several analogues examined, [Gln(6)]alpha-melanocyte-stimulating hormone (MSH)-ND was found to have 10000 times less biological activity than alpha-MSH-ND for the MC1R, whereas, the potencies of both oligopeptides were comparable in both the melanocortin-3 receptor (MC3R) and MC4R. [Gln(6)]alpha-MSH-ND exhibited a type I' beta-turn that was similar to the type I beta-turn structure of alpha-MSH-ND. However, a remarkable structural difference was observed with respect to the side chain orientations of the sixth and seventh residues of [Gln(6)]alpha-MSH-ND, which were found to be mirror images of alpha-MSH-ND. By homology modeling analysis, the His(6) of alpha-MSH-ND was found to interact with the TM2 regions of all three receptors (Glu(94) of MC1R, Glu(94) of MC3R, and Glu(100) of MC4R), but [Gln(6)]alpha-MSH-ND did not. The phenyl ring of the D-Phe(7) residue of [Gln(6)]alpha-MSH-ND revealed an interaction with the TM3 regions of both the MC3R and MC4R (Ser(122) of MC3R or Ser(127) of MC4R). However, in the MC1R, these serine residues corresponded to Val(122), which contains two methyl groups that induce steric hindrance with D-Phe(7) of [Gln(6)]alpha-MSH-ND. This is a possible explanation for the biological activity of [Gln(6)]alpha-MSH-ND for the MC1R being significantly lower than that for either the MC3R or MC4R. CONCLUSIONS: Minimization of the MC1R selectivity whilst preserving its comparable potency for both the MC3R and MC4R could be achieved by modifying the D-Phe(7) orientation of alpha-MSH-ND, while maintaining the 'type I beta-turn'-like structure.

Animals↗

PubMed2001

Precursor RNAs harboring nonsense codons accumulate near the site of transcription.

Messenger RNAs containing premature termination codons (PTCs) are selectively eliminated by nonsense-mediated mRNA decay (NMD). Paradoxically, although cytoplasmic ribosomes are the only known species capable of PTC recognition, in mammals many PTC-containing mRNAs are apparently eliminated prior to release from the nucleus. To determine whether PTCs can influence events within the nucleus proper, we studied the immunoglobulin (Ig)-mu and T cell receptor (TCR)-beta genes using fluorescent in situ hybridization (FISH). Alleles containing PTCs, but not those containing a missense mutation or a frameshift followed by frame-correcting mutations, exhibited elevated levels of pre-mRNA, which accumulated at or near the site of transcription. Our data indicate that mRNA reading frame can influence events at or near the site of gene transcription.

Animals↗

PubMed2001

Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase.

The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.

Animals↗

PubMed2001

Inhibition of the transcription factors AP-1 and NF-kappaB in CD4 T cells by peroxisome proliferator-activated receptor gamma ligands.

The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, is essential for adipocyte differentiation and glucose homeostasis. PPARgamma has been found recently to regulate macrophage activation in response to mitogens and inflammation. Our study shows PPARgamma to be preferentially expressed in the nuclei of resting T cells and to increase upon activation of T cells by either anti-CD3 and anti-CD28 or phorbol myristyl acetate (PMA). We also found the PPARgamma ligand ciglitizone to attenuate the activation of T cells by inhibiting cytokine gene expression and anti-CD3 and anti-CD28 or PMA-induced proliferative responses. Inhibition of both the proliferative response and inflammatory cytokine expression in CD4 T cells was correlated with suppression of the activated transcription factors AP1 and NF-kappaB. PPARgamma ligands also strongly inhibited SEA-induced Vbeta3 T cell activation in vivo. These results, together with previous findings of the inhibitory effect of PPARgamma ligands on activated macrophages, provide clear evidence for PPARgamma as a negative regulator of the inflammatory activation of both macrophage and T cells. PPARgamma may thus be a potential therapeutic target for the treatment of autoimmunity.

Animals↗

PubMed2001

Up-converting phosphor reporters for nucleic acid microarrays.

An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.

Biotinylation↗

PubMed2001

Primary cutaneous T-cell-rich B-cell lymphoma: clinically distinct from its nodal counterpart?

The cases of two patients with Stage IE primary cutaneous T-cell-rich B-cell lymphoma (TCRBCL) are described. In both, the lesion showed a dense infiltrate by numerous small T lymphocytes with scattered histiocytes and large atypical B-lymphoid cells. Polymerase chain reaction assays demonstrated that the B cells were monoclonal, with immunoglobulin heavy-chain gene rearrangement. No clonal rearrangements of the T-cell receptor gamma gene were observed. Both patients were disease-free at 4 months and at 5 years after therapy, respectively. Although rare, primary cutaneous T-cell-rich B-cell lymphoma appears to have a better prognosis than its nodal counterpart, with or without skin involvement.

Antigens, CD20↗

PubMed2001

Solution structure of Ca(2+)-calmodulin reveals flexible hand-like properties of its domains.

The solution structure of Ca(2+)-ligated calmodulin is determined from residual dipolar couplings measured in a liquid crystalline medium and from a large number of heteronuclear J couplings for defining side chains. Although the C-terminal domain solution structure is similar to the X-ray crystal structure, the EF hands of the N-terminal domain are considerably less open. The substantial differences in interhelical angles correspond to negligible changes in short interproton distances and, therefore, cannot be identified by comparison of NOEs and X-ray data. NOE analysis, however, excludes a two-state equilibrium in which the closed apo conformation is partially populated in the Ca(2+)-ligated state. The difference between the crystal and solution structures of Ca(2+)-calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets. In contrast, the vast majority of side chains making up the target binding surface are locked into the same chi(1) rotameric states as in complexes with target peptide.

Animals↗

PubMed2001

Intramuscular electroporation delivery of IL-12 gene for treatment of squamous cell carcinoma located at distant site.

Gene therapy with IL-12 has been shown to elicit potent systemic antitumor response in a variety of tumors. Although direct intratumoral injection is the most commonly used delivery route for gene therapy of solid tumors, the skeletal muscle has been shown to be an ideal tissue for gene delivery to produce systemic gene expression. We have previously demonstrated that electroporation delivery of a reporter gene to muscle enhances the transfection efficiency and the level of gene expression by two to three logs. We report here that intramuscular (i.m.) injection of as little as 10 microg of the IL-12 DNA plasmid followed by electroporation prevents squamous cell carcinoma (SCCVII) tumor establishment in up to 40% of experimental animals and reduces the volume of established tumors by 75% compared to controls (P<.05). By comparison, there was no difference in tumor growth observed between IL-12 injection alone and injection of empty vector with or without electroporation. The induction of antitumor activity by i.m. electroporation delivery of the IL-12 gene is associated with an increase in IL-12 expression in muscle and serum. The level of IL-12 expression in muscle and serum was 1500 pg/tibialias muscle and 170 pg/mL serum, respectively, at day 6, after the gene was delivered by electroporation. In contrast, the level of IL-12 when the gene was injected without electroporation was hardly detectable after subtracting the background level of IL-12 detected in naïve mice. The high level of IL-12 expression led to a 170-fold induction of IFN-gamma expression in serum at day 6 after i.m. electroporation delivery of IL-12 DNA plasmid, which was equal to 1450 pg/mL in the serum. The induction of antitumor activity by i.m. electroporation delivery of the IL-12 gene also correlates with increased CD8+ T-cell population in peripheral blood but not in spleen. Our findings suggest that i.m. delivery of IL-12 gene using electroporation is an effective method of inducing a systemic antitumor response against SCC.

Animals↗

PubMed2001

Efficient in vivo gene transfer by PCR amplified fragment with reduced inflammatory activity.

There are many problems associated with plasmid DNA that may limit its use in systemic gene transfer. These problems could be solved by the use of synthetic genes. As a model to test the feasibility of using synthetic genes for gene therapy, we PCR-amplified a fragment containing the CMV promoter, the luciferase gene and a polyadenylation signal. The in vivo expression efficiency of the PCR fragment was determined by using two different methods, a hydrodynamics-based gene transfer of naked DNA to the liver and LPD (a lipid-based vector) mediated gene transfer to the lung. Our results show that linear fragments are at least as active as plasmid DNA following systemic delivery by LPD. However, PCR fragments are much less inflammatory than plasmid DNA as shown by a three-fold reduction in serum levels of both TNF-alpha and IL-12. Our results also showed that PCR fragments are highly efficient in liver gene transfer following systemic administration in a large volume. Thus, these results support the idea of using synthetic genes for gene therapy. Since gene sequence can be easily obtained as a PCR fragment, our results also imply that it may provide a useful and convenient method for determining the physiologic function of a putative gene in intact animals.

Animals↗

PubMed2001

Intramuscular electroporation delivery of IFN-alpha gene therapy for inhibition of tumor growth located at a distant site.

Although electroporation has been shown in recent years to be a powerful method for delivering genes to muscle, no gene therapy via electro-injection has been studied for the treatment of tumors. In an immunocompetent tumor-bearing murine model, we have found that delivery of a low dose of reporter gene DNA (10 microg) to muscle via electroporation under specific pulse conditions (two 25-ms pulses of 375 V/cm) increased the level of gene expression by two logs of magnitude. Moreover, administration of 10 microg of interferon (IFN)-alpha DNA plasmid using these parameters once a week for 3 weeks increased the survival time and reduced squamous cell carcinoma (SCC) growth at a distant site in the C3H/HeJ-immunocompetent mouse. IFN-alpha gene therapy delivered to muscle using electroporation demonstrated statistically significant (P < 0.05) therapeutic efficacy for treating SCC located at a distant site, compared with interleukin (IL)-2 or endostatin gene, also delivered by electro-injection. The increased therapeutic efficacy was associated with a high level and extended duration of IFN-alpha expression in muscle and serum. We also discovered that the high level of IFN-alpha expression correlated with increased expression levels of the antiangiogenic genes IP-10 and Mig in local tumor tissue, which may have led to the reduction of blood vessels observed at the local tumor site. Delivery of increasing doses (10-100 microg) of IFN-alpha plasmid DNA by injection alone did not increase antitumor activity, whereas electroporation delivery of increasing doses (10-40 microg) of IFN-alpha plasmid DNA did increase the survival time. Our data clearly demonstrate the potential utility of electroporation for delivery of gene therapy to muscle for the treatment of residual or disseminated tumors.

Animals↗

PubMed2001

Muscle-specific enhancement of gene expression by incorporation of SV40 enhancer in the expression plasmid.

Skeletal muscle is established as an ideal tissue for gene delivery to treat systemic diseases. However, the relatively low levels of gene expression obtained from using naturally occurring promoters, including the strong cytomegalovirus (CMV) enhancer/promoter (E/P), have limited the use of muscle as a target tissue. The relatively weak simian virus 40 (SV40) enhancer is known to have dual functions promoting localization of DNA to the nucleus and activating transcription. An SV40 enhancer incorporated either at the 5' end of CMV E/P or the 3' end of the polyadenylation site gave as much as a 20-fold increase in the level of exogenous gene expression in muscle in vivo, compared with expression observed with CMV E/P alone. The minimum requirement for this enhancement is a single copy of a 72-bp element of the SV40 enhancer, in combination with either the CMV E/P or skeletal actin (SkA) promoter. Enhancement of gene expression in muscle by this SV40 enhancer was also observed by using the powerful electroporation delivery. However, the SV40 enhancer does not increase the level of CMV E/P driven reporter gene expression in dividing tumor cells in vivo and in the dividing myoblast cell C2C12 in vitro. The data suggest that including this enhancer in the plasmid will enhance the level of gene production for muscle-based gene therapy.

Animals↗

PubMed2001

The inositol 5-phosphatase SHIP is expressed as 145 and 135 kDa proteins in blood and bone marrow cells in vivo, whereas carboxyl-truncated forms of SHIP are generated by proteolytic cleavage in vitro.

The inositol polyphosphate 5-phosphatase SHIP plays an important role in negative signalling in B cells and mast cells and in the down-regulation of cytokine receptor-mediated signals in myeloid cells. SHIP is expressed as a 145 kDa full-length protein and an isoform of 135 kDa due to alternative splicing. Additional smaller forms of SHIP which are truncated at the carboxy terminus have been described in bone marrow and peripheral blood mononuclear cells (PBMC). Our data demonstrate that human bone marrow cells and PBMC from healthy donors and patients with acute myeloid leukemia express the 145 kDa form of SHIP and low amounts of a 135 kDa form of SHIP in vivo whereas C-terminal-truncated SHIP proteins are generated by a PMSF-sensitive protease during the preparation of cell lysates in vitro. We have further characterized this protease and identified a proteolytic cleavage site in the human SHIP protein C-terminal to tryptophan residue 941. These data support a physiological role for the 145 and 135 kDa forms of SHIP in bone marrow and peripheral blood cells from normal donors and patients with acute myeloid leukemia.

Alternative Splicing↗

PubMed2001

Chemiluminescence of Ce(IV) and surfactant Tween 20.

It was found that cerium(IV) reacted with surfactant Tween 20 in acidic medium to generate green chemiluminescence. The maximum emission wavelength was about 478 nm. The enhancement and inhibition of the CL reaction by some polyphenols and anilines was studied and the possibility of analytical application was explored.

Journal Article↗

PubMed2001

Separation and preconcentration of chromium species by selective absorption on Lemna minor and determination by slurry atomisation electrothermal atomic absorption spectrometry.

A novel method for the separation and preconcentration of Cr(III)/Cr(VI) with Lemna minor and determination by slurry atomization electrothermal atomic absorption spectrometry (ETAAS) was developed. A sample solution was added to a polyethylene beaker containing 10 mg of 160 mesh pre-treated Lemna minor, adjusted to pH 1.0, stirred for 8 min for selective absorption of Cr(III) and then centrifuged. The upper layer of solution was transferred into another polyethylene beaker containing 10 mg of 160 mesh pre-treated Lemna minor, adjusted to pH 5.0, stirred for 12 min for adsorption of the residual Cr(VI) and centrifuged. The two residues in two centrifuge tubes were washed twice with water, 2 ml of agar solution added, stirred for 2 min, then two slurries were prepared and used for the determination of Cr(III) and Cr(VI) by ETAAS. Detection limits (3sigma) of 0.01 microg L(-1) for Cr(III) and 0.03 microg L(-1) for Cr(VI) were obtained. The relative standard deviation was 2.8% for Cr(III) and 3.3% for Cr(VI) at the 1 microg L(-1) level. The method was applied to the determination of Cr(III)/Cr(VI) in water samples. The analytical recoveries of Cr(III) and Cr(VI) added to samples were 97-102 and 96-103%, respectively.

Journal Article↗

PubMed2001

Characterization of genes expressed in the salivary glands of the tsetse fly, Glossina morsitans morsitans.

Salivary gland products of haematophogous insects including tsetse flies (Diptera: Glossinidia) are involved in antihaemostasis to allow for efficient blood feeding. In addition, salivary products of tsetse are thought to indirectly support the metacyclogenesis and eventual transmission of the African trypanosome protozoan parasites to their mammalian hosts. We have previously characterized the major anticoagulant, Tsetse Thrombin Inhibitor (TTI), from salivary extracts, and described molecular aspects of its cDNA from a Glossina morsitans morsitans salivary gland cDNA library. In addition, a family of two related genes with growth factor and adenosine-deaminase motifs (TSGF-1 and TSGF-2) have also been described. Here, we report on the molecular aspects of three different cDNAs and their putative products expressed in salivary glands: cDNAs TAg5, Tsal1 and Tsal2. The full-length transcript encoded by Tsetse Antigen 5 (TAg5) cDNA is 926 bp excluding the poly(A) stretch, and has an open reading frame of 259 amino acids that can encode for a protein of 28 925 Da. The putative product of TAg5 shows extensive similarities to cDNAs characterized from Drosophila (Agr and Agr2) and sandfly Lutzomyia (LuLoAG5). The cDNAs Tsal1 and Tsal2 are predicted to encode for mature proteins of 45 612 Da (399 amino acids) and 43 930 Da (389 amino acids), respectively, and their putative products exhibit over 42% identity to one another. The N terminus of each putative protein contains a hydrophobic region with signal peptide characteristics indicating that they may be secretory in nature. Transcripts specific for TAg5 and Tsal2 genes can be detected in all developmental stages of tsetse while Tsal1 expression is limited to adult and larval stages. A reverse transcription polymerase chain reaction based amplification approach indicates that TAg5 transcipts can be detected from proventriculus and midgut tissues of the fly in addition to salivary glands, while Tsal1 and Tsal2 expression is restricted to salivary gland and proventriculus. The salivary glands of adult males are found to express higher levels of TAg5 and Tsal2 in comparison to females while no significant sex-based difference is observed for Tsal1 expression. The expression of these cDNAs in different tsetse species (G. m. morsitans, Glossina austeni and Glossina fuscipes) shows wide variations.

Amino Acid Sequence↗

PubMed2001

Obesity in adults and children: a call for action.

Obesity/overweight in adults and children is a worldwide health problem associated with substantial economic burden as measured by paid sick leave, life and disability insurance rates, and obesity-related physician visits and hospital stays. Overweight/obese people experience hypertension, elevated cholesterol, and type 2 diabetes and suffer more joint and mobility problems than people within the normal weight for height range. While there is need to understand individual behaviors that can be modified to promote weight loss and weight maintenance, there is as great a need to consider contextual factors at the societal level that can impede or even sabotage weight control efforts. In every country with improved living standards people will continue to eat too much and engage in too little physical activity. The call for action is for all modernized societies to alter environments and attitudes to support, rather than hinder, healthy dietary intake and being physically active.

Absenteeism↗

PubMed2001

Role of extra-ovarian oestrogens in the regulation of gonadotropin releasing hormone mRNA expression in the rat brain.

To further understand the role of oestrogens in the regulation of gonadotropin releasing hormone (GnRH) mRNA expression in the female rat brain, the effect of EM-652.HCl, a pure anti-oestrogen, was studied in intact and ovariectomized rats as well as in rats chronically treated with a GnRH agonist D-trp6, des-Gly-NH210 GnRH ethylamide (GnRH-A), a treatment which blocks ovarian steroidogenesis. Quantitative in situ hybridization was used to measure GnRH mRNA at the cellular level in the preoptic-anterior hypothalamic area. It was found that, 49 weeks after ovariectomy (OVX), the number of silver grains per cell corresponding to GnRH mRNA was increased by 34%. Long-term administration (49 weeks) of EM-652.HCl to OVX rats resulted in a further increase (11% over the levels measured in OVX rats) in the hybridization signal. By contrast, in intact female rats, treated during 52 weeks with EM-652.HCl, a 49% increase in the GnRH hybridization signal was detected. In rats treated with GnRH-A during the same period, a 20% decrease in GnRH mRNA was observed. When EM-652.HCl was administered concomitantly with GnRH-A, a further 63% increase over the mRNA levels recorded in GnRH-A treated rats was found. Thus, long-term treatment with the anti-oestrogen EM-652.HCl can upregulate GnRH mRNA expression in intact female rats, OVX rats and female rats chronically treated with a GnRH-A. It is suggested that the pure anti-oestrogen EM-652.HCl can exert an influence on the oestrogen feedback mechanism involved in the regulation of GnRH neuronal activity by neutralizing the action of locally produced or low circulating levels of oestrogens remaining after OVX or GnRH-A treatment.

Animals↗

PubMed2001

Role of vicinal cysteine pairs in metalloid sensing by the ArsD As(III)-responsive repressor.

ArsD is a 120-residue repressor that regulates expression of the arsRDABC arsenical resistance operon of plasmid R773 in Escherichia coli. ArsD is released from arsRDABC promoter DNA by binding of the compounds with the metalloids As(III) or Sb(III). ArsD has three vicinal cysteine pairs, Cys-12 and Cys-13, Cys-112 and Cys-113 and Cys-119 and Cys-120. In this study, the role of these three cysteine pairs was investigated. Mutation or deletion of Cys-119-Cys-120 had no effect on repression or metalloid responsiveness in vivo or in vitro. Mutagenesis of either the Cys-12-Cys-13 pair or the Cys-112-Cys-113 pair had no effect on repression but produced loss of inducibility, suggesting that both Cys-12-Cys-13 and Cys-112-Cys-113 may be required for As(III) or Sb(III) responsiveness. Assays of binding of wild-type and mutant ArsDs by As(III) affinity chromatography showed that each of the three vicinal cysteine pairs is capable of binding As(III) independently. The effect of As(III) or Sb(III) on intrinsic protein fluorescence was used to examine the properties of individual cysteine pairs. The fluorescence of Trp-97 was shown to be quenched by the addition of Sb(III) or As(III). The vicinal Cys-112-Cys-113 pair was required for the majority of the metalloid-dependent quenching of Trp-97 fluorescence. The data are consistent with a model in which Cys-12-Cys-13 and Cys-112-Cys-113 form independent As(III) binding sites, both of which are required for in vivo ArsD function.

Amino Acid Sequence↗