Biomedical subjects

S Li

Publications and source records attributed to S Li.

At least 1,189 records · Page 66Linked to original sources

PubMed1996

Toxicity of ochratoxin A, its opened lactone form and several of its analogs: structure-activity relationships.

Ochratoxin A (OA); its three natural analogs, ochratoxin C (OC), B (OB), and alpha (Oalpha); and its six synthetic analogs, the epimere of OA (d-OA), the ethylamide of OA (OE-OA), decarboxylated OA (DC-OA), O-methylated OA (OM-OA), lactone-opened OA (OP-OA), and the methyl ester of Oalpha (M-Oalpha) were assayed for their toxicities in prokaryotic (Bacillus brevis) and eukaryotic (HeLa cell) systems and in animals (mouse and rat). The LC50S (mM) for HeLa cells, were 0.005 (OA), 0.009 (OC), 0.163 (d-OA), 10.1 (OE-OA), 7.6 (DC-OA), 0.83 (OM-OA), 0.054 (OB), and 0.56 Oalpha). The minimum inhibitory doses (nmol/disc) for the growth of B. brevis (pH 6.5) were 8.7 (OA), 2.0 (OC), 5.5 (d-OA), 1.1 (OE-OA), 54 (OB), 390 (Oalpha), and 90 (M-Oalpha) while no inhibition of the bacterial growth was observed for OM-OA, DC-OA, and OP-OA at doses as high as 350 nmol/disc. The results indicate that the toxicities of OA were associated with its isocoumarin moiety but that neither the dissociation of the phenolic hydroxyl group nor the iron-chelating properties of OA were directly related to its toxicities. The lactone carbonyl group of OA, however, appears to be involved in OA toxicity as OP-OA is found in the bile of rats injected with OA and has similar toxicity to that of OA when administered intravenously to the rat. Overall, the structure-activity studies suggest that the toxicity of OA is attributable to its isocoumarin moiety and that the lactone carbonyl group may be involved in its toxicity.

Animals↗

PubMed1996

Localization of gamma-glutamylcysteine synthetase mRNA expression in mouse brain following methylmercury treatment using reverse transcription in situ PCR amplification.

In previous studies we reported that prolonged treatment of rats with subtoxic levels of mercury as methymercury hydroxide (MMH) elicited a two- to three-fold increase in renal glutathione (GSH) content and a three- to fourfold increase in the mRNA encoding the catalytically active heavy subunit of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Since methylmercury is a potent neurotoxicant, we investigated the effect of methylmercury treatment on GSH synthesis and the distribution of GCS mRNA expression in the brain. Male C57B1/ 6 mice were treated for 3 consecutive days with MMH (3 mg/kg/ day,i.p.). GSH levels in whole brains were increased by twofold 24 hr following the first injection and remained elevated two to three times control levels after two subsequent MMH treatments. Concomitantly, whole brain GCS mRNA levels were increased 2.7-fold 24 hr after the third MMH treatment. Reverse transcription in situ PCR amplification of GCS heavy subunit mRNA in brain slices taken from MMH-treated mice showed that GCS expression was selectively localized to the cerebellum and hippocampal regions and, within these regions, to areas which are known to resist methylmercury toxicity. In contrast, no GCS mRNA expression was found in brain regions which are known to be highly susceptible to mercury toxicity. These findings suggest that resistance to methylmercury toxicity in the brain may reflect the ability of specific neuronal cell types to up-regulate GSH synthesis as a protective response to mercury-mediated cell damage.

Animals↗

PubMed1996

Effects of vomitoxin (deoxynivalenol) on transcription factor NF-kappa B/Rel binding activity in murine EL-4 thymoma and primary CD4+ T cells.

The trichothecene mycotoxin vomitoxin (VT, deoxynivalenol) superinduces the gene expression of IL-2 and several other cytokines in both cellular and murine models. Because transcription factor NF-kappaB/Rel has been shown to play a crucial role in control of cytokine gene transcription, we assessed the in vitro effects of VT on NF-kappaB/Rel binding activity by electrophoretic mobility shift assay using both cloned (EL-4) and primary (CD4+) murine T cells. When EL-4 thymoma cells were stimulated with phorbol 12-myristate 13-acetate plus ionomycin in the presence of 500 ng/ml VT, DNA binding activity by NF-kappaB/Rel in nuclear extracts was increased from 2 to 48 hr when compared to controls employing no VT. VT preferentially induced a slower migrating electrophoretic band of the NF-kappaB/Rel complex particularly in later time points (8-48 hr). The band was found to contain a c-Rel/p50 heterodimer by supershift assay using antibodies specific for c-Rel and p50. NF-kappaB/Rel binding activity was enhanced by VT in a dose-dependent fashion. As little as 50 ng/ml VT was sufficient to increase NF-kappaB/Rel binding in a 1-hr EL-4 culture. Using Western blot analysis, effects on EL-4 cells were further related to VT-mediated inhibition of resynthesis of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB/Rel. Decreased IkappaBalpha levels were observed with 250-1000 ng/ml VT from 4 to 48 hr. Using primary murine CD4+ T cell cultures, elevated NF-kappaB/Rel and c-Rel/p50 binding activities were also observed at VT of 500 ng/ml from 1 to 72 hr concurrently with decreased IkappaBalpha levels. These data suggest that VT increased NF-kappaB/Rel binding activity and, in particular, the transactivating form, c-Rel. Increased NF-kappaB/Rel binding activity in the later (48 hr) stages of cell incubation may be explained, in part, by VT-mediated inhibition of resynthesis of its cytoplasmic inhibitor IkappaBalpha and by decreases in the inhibitory p50 homodimer. Elevated NF-kappaB/Rel binding activity may be involved mechanistically in VT-induced gene expression of the cytokines and resultant toxic and autoimmune effects.

Animals↗

PubMed1996

Localization by in situ hybridization of gamma-glutamylcysteine synthetase mRNA expression in rat kidney following acute methylmercury treatment.

In previous studies we reported that prolonged treatment of rats with subtoxic levels of mercury as methylmercury hydroxide (MMH) elicited a two- to threefold increase in renal glutathione (GSH) content and a three- to fourfold increase in the mRNA encoding the catalytically active heavy subunit of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. In the present studies, we demonstrate that enhanced expression of GCS mRNA and GSH synthesis rapidly occur following acute MMH treatment and, moreover, that increased expression of renal GCS mRNA is localized predominantly to regions of the kidney cortex consistent with the principal distribution of mercury in the kidney. Previous studies have demonstrated that resistance to mercury toxicity during prolonged MMH exposure may be associated with the ability to up-regulate GSH synthesis subsequent to intracellular dealkylation of MMH to Hg2+. The present finding that GCS mRNA and GSH levels are rapidly increased in kidney cells which are most susceptible to mercury toxicity supports the view that up-regulation of GSH synthesis occurs as an initial adaptive response to Hg2(+)-mediated cytotoxicity following acute as well as prolonged mercury exposure.

Animals↗

PubMed1996

The myosin filament XV assembly: contributions of 195 residue segments of the myosin rod and the eight C-terminal residues.

A mixture of two peptides of approximately M(r) 13000 has been isolated from a papain digest of LC2 deficient myosin. The peptides assemble into highly ordered aggregates which in one view are made up of strands of pairs of dots with an average side to side spacing of 13.0 nm and an average axial repeat of 9.0 nm. In another view there are strands of single dots with a side-to-side spacing of 7.8 nm and an axial repeat of 9.1 nm. From N-terminal peptide sequencing, the two peptides have been shown to come from regions of the myosin rod displaced by 195 residues. We have shown that either peptide alone can assemble to form the same aggregates. The 195 residue displacement of the M(r) 13000 peptides corresponds closely to the 196 residue repeat of charges along the myosin rod. This finding permits us to designate 195 residue segments of the myosin rod and to relate assembly characteristics directly to the similar 195 residue segments and 196 residue charge repeat. The most C-terminal 195 residue segment carries information for assembly into helical strands. The contiguous 195 residue segment, in major part, carries information for the unipolar assembly, characteristic of the assembly in each half of the myosin filament. The next contiguous 195 residue segment, in major part, carries information for bipolar assembly which is characteristic of the bare zone region of the filament; and for the transition from the bipolar bare zone to unipolar assembly. The effect of the eight C-terminal residues of the myosin rod on the assembly of the contiguous 195 residues has also been studied. The entire fragment of 195 + eight C-terminal residues assembled to form helical strands with an axial repeat of 30 nm. Successive deletion of charged residues changed the axial repeat of the helical strands suggesting that the charged residues at the C-terminus are involved in determining the pitch in the helical assembly of the contiguous 195 residues.

Amino Acid Sequence↗

PubMed1996

Lower 3H-paroxetine binding in cerebral cortex of suicide victims is partly due to fewer high affinity, non-transporter sites.

Suicide has been associated with decreased serotonin transmission. Measurement of concentrations of serotonin, its precursors tryptophan (TRY) and 5-hydroxytryptophan (5-HTP) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), have been used as indices of serotonin activity, and with serotonin transporter binding, are indices of the integrity of serotonin nerve terminals. Most previous studies have not distinguished high affinity transporter binding from a very similar nontransporter binding site, where binding is not dependent on Na+ or Cl- and that does not have a known functional role. We therefore, assayed binding kinetics in prefrontal (PFC) and temporal cortex (TC) in matched pairs of suicide victims and controls using the selective ligand 3H-paroxetine, and employing 1 microM sertraline to define specific binding to the transporter and 10 microM sertraline which also displaces binding to the high affinity, nontransporter site. In addition, we measured concentrations of TRY, 5-HTP, serotonin and 5-HIAA in the same brain areas. The total number of 3H-paroxetine transporter and nontransporter binding sites (Bmax), was lower in the suicide group compared to controls in both Brodmann area 9 (prefrontal cortex; p = 0.02) and in Brodmann area 38 (temporal cortex, p = 0.01). In contrast, no differences were found in the number of high affinity transporter binding sites and concentrations of serotonin, 5-HIAA, 5-HTP or TRY (p > 0.05). We conclude that the number of serotonin transporter sites is not altered in Brodmann area 9 in suicide, and that fewer 3H-paroxetine and 3H-imipramine binding sites found in this region of cerebral cortex of suicides may be explained by a reduction in the nontransporter binding sites.

5-Hydroxytryptophan↗

PubMed1996

In vitro and in vivo studies of three radiolabelled somatostatin analogues: 123I-octreotide (OCT), 123I-Tyr-3-OCT and 111In-DTPA-D-Phe-1-OCT.

Scintigraphy with long-acting somatostatin (SST) analogues may be useful for the localization of tumours expressing receptors (R) for SST. In this study we have analysed the in vitro and in vivo binding properties of three SST analogues, 123I-octreotide (OCT), 123I-Tyr-3-OCT and 111In-DTPA-d-Phe-1-OCT. In vitro binding studies performed with a variety of primary tumours (n=48) as well as with several tumour cell lines (A431, HT29, PANC1, COLO320, HMC1, KU812) indicated significant in vitro binding of these three radiolabelled SST analogues to two subpopulations of SSTR, high (Kd 0.2-2.0 nM) and low (Kd 5-15 nM) affinity ones. The number of SSTR on tumour cells was at least a 1000-fold higher as compared with normal peripheral blood cells. Comparative scintigraphic studies using 123I-OCT and/or 123I-Tyr-3-OCT and/or 111In-DTPA-d-Phe-1-OCT were performed in 21 patients with histologically verified intestinal carcinoid tumours. Corresponding scintigraphic results were obtained in 18 of 21 patients investigated with two different SSTR ligands, either 123I-OCT/123I-Tyr-3-OCT (four of five), 123I-OCT/111In-DTPA-d-Phe-1-OCT (eight of nine), or 123I-Tyr-3-OCT/111In-DTPA-d-Phe-1-OCT (six of seven). We conclude that various tumours express high amounts of SSTR which are recognized by three radiolabelled SST analogues: 123I-OCT, 123I-Tyr-3-OCT and 111In-DTPA-d-Phe-1-OCT. Differences between these SST analogues in their in vitro binding and/or in vivo scanning properties are observed in a minority of patients. Thus, the labelling of OCT with iodine may be an alternative approach for those nuclear medicine departments for which 111In-DTPA-d-Phe-1-OCT is not easily available, or is too expensive.

Adolescent↗

PubMed1996

Closed anesthesia with nitrous oxide.

Anesthesia was induced with intravenous thiopental, fentanyl, and succinylcholine, and maintained with isoflurane, N2O-O2 and atracurium after denitrogenation in 20 adult patients. Inspired and end-tidal concentrations of O2, N2O, CO2, isoflurane and oxygen saturation of pulse oximeter (SpO2) were monitored. After intubation, N2O (750 ml/min) and O2 (250 ml/min) were administrated. When inspired N2O concentration (FiN2O) reached 60% the closed anesthesia was initiated by adjusting fresh gas flow rate in order to meet the oxygen demand of the patients. The N2O/O2 ratio was constantly kept at 0.7. The results revealed that during wash-in period, FiN2O increased, on average, to 30% at 3.83 min, 40% at 6.22 min, 50% at 14.13 min respectively. After 30 min of closed anesthesia, FiN2O decreased to 49.4%. It was 46.8% at 60 min. Thereafter it increased smoothly to 51.9% at 180 min. No hypoxia and hypercapnia occurred. Compared with commonly used high flow N2O-N2 inhalation, the technique of low flow wash-in and closed circuit maintenance with N2O-O2 may be less expensive and feasible and causes less pollution.

Abdomen↗

PubMed1996

Studies on the ossification of compound transplantation of porous TCP ceramics and bone marrow.

Following the culture of human red marrow with porous TCP ceramics in vitro, a large amount culturing marrow cells adhered well to the surface and inner wall of TCP ceramics and spread well over the dish bottom 4 weeks after cultivation. After the compound transplantation of porous TCP ceramics with red marrow into quadriceps femoris muscle of rabbits, a huge amount of new bone and marrow formed in these sites on and in the ceramics 4 weeks after transplantation. These results showed an excellent biocompatibility between TCP ceramics and marrow and an excellent osteogenic effect in the compound transplantation. The mechanism of ossification and clinical significance about the compound transplantation were discussed.

Animals↗

PubMed1996

Effects of temperature and white sucker (Catostomus commersoni) serum supplement on the in vitro multiplication of Cryptobia catostomi in cell-free culture medium.

Cryptobia catostomi, a non-pathogenic haemoflagellate of white suckers (Catostomus commersoni) multiplied rapidly in modified TDL-15 medium supplemented with 10% fetal bovine serum and 1% heatinactivated white sucker serum (WSS) at 10 degrees C and 18 degrees C. The numbers of C. catostomi counted were significantly higher in cultures incubated at 10 degrees C than in those incubated at 18 degrees C beginning at 3 weeks post-incubation. The culture forms (from the eighth subculture at 9 months after isolation) were morphologically similar to blood forms and were infective to laboratory-raised white suckers. The parasite survived for about 4 weeks in the medium without WSS at 18 degrees C. The present study indicates that WSS supplement supports the in vitro multiplication of C. catostomi and that its multiplication is temperature-dependent.

Animals↗

PubMed1996

Norepinephrine levels in discrete brain nuclei in borderline hypertensive rats exposed to compound stressors.

The borderline hypertensive rat (BHR) appears to be an appropriate model for investigating the role of the environment in producing hypertension. Previous studies have demonstrated that the BHR shows chronic blood pressure elevations to both stress and high salt intake. Other studies suggest that interactions between the brain and kidney play an important role in initiating this hypertension. The central noradrenergic system has been implicated in these effects, especially in the hypothalamus. Because exercise has been found to attenuate stress-induced hypertension in the BHR, the current study sought to examine the impact of stressors paired with exercise (salt intake or stress) with those combining stress and high salt. Male BHR were exposed to either control, salt plus stress, salt plus exercise, or stress plus exercise conditions for either 2 or 6 months, beginning at 2 months of age. Following sacrifice, brain nuclei in the brain stem and hypothalamus were removed using the Palkovits micropunch technique. Punches were analyzed for NE content via liquid chromatography with electrochemical detection. Compared with the control condition, chronic salt plus stress led to reductions in NE content, especially in the hypothalamus. Compared with salt plus stress, the exercise conditions were associated with elevated NE levels, especially in the early phases of exposure to the treatment. The possible role of exercise training in preventing a central nervous system trigger from inducing hypertension in the BHR is discussed.

Animals↗

PubMed1996

Effects of alcohols on the phase transition temperatures of mixed-chain phosphatidylcholines.

The biphasic effect of ethanol on the main phase transition temperature (Tm) of identical-chain phosphatidyl-cholines (PCs) in excess H2O is now well known. This biphasic effect can be attributed to the transformation of the lipid bilayer, induced by high concentrations of ethanol, from the partially interdigitated L beta, phase to the fully interdigitated L beta I phase at T < Tm. The basic packing unit of the L beta I phase has been identified recently as a binary mixture of PC/ethanol at the molar ratio of 1:2. The ethanol effect on mixed-chain PCs, however, is not known. We have thus in this study investigated the alcohol effects on the Tm of mixed-chain PCs with different delta C values, where delta C is the effective acyl chain length difference between the sn-1 and sn-2 acyl chains. Initially, molecular mechanics (MM) simulations are employed to calculate the steric energies associated with a homologous series of mixed-chain PCs packed in the partially and the fully interdigitated L beta I motifs. Based on the energetics, the preference of each mixed-chain PC for packing between these two different motifs can be estimated. Guided by MM results, high-resolution differential scanning calorimetry is subsequently employed to determine the Tm values for aqueous lipid dispersions prepared individually from a series of mixed-chain PCs (delta C = 0.5-6.5 C-C bond lengths) in the presence of various concentrations of ethanol. Results indicate that aqueous dispersions prepared from mixed-chain PCs with a delta C value of less than 4 exhibit a biphasic profile in the plot of Tm versus ethanol concentration. In contrast, highly asymmetric PCs (delta C > 4) do not exhibit such biphasic behavior. In the presence of a longer chain n-alcohol, however, aqueous dispersions of highly asymmetric C(12):C(20)PC (delta C = 6.5) do show such biphasic behavior against ethanol. Our results suggest that the delta C region in a highly asymmetric PC packed in the L beta I phase is most likely the binding site for n-alcohol.

Alcohols↗

PubMed1996

Inhibitory effect of sugars and polyols on the metal-catalyzed oxidation of human relaxin.

Previously, our laboratory (Li, S.; et al. Biochemistry 1995, 34, 5762-5772) showed that the oxidation of recombinant human relaxin (Rix) could be induced by ascorbic acid (AsA)/Cu(II), a system used for the metal-catalyzed generation of reactive oxygen species. In this study, we observed that this oxidation could be inhibited by high concentrations of mannitol and other sugars and polyols, such as ethylene glycol, glycerol, glucose, and dextran. Similar protective effects with high concentrations of mannitol were also observed in the AsA/CuCl2-induced oxidation of Gly-Met-Gly and Gly-His-Gly. In contrast, (carboxymethyl)cellulose had no protective effect on the metal-catalyzed oxidation of Rix. These results, together with results from deuterium isotope experiments and spectroscopic experiments, suggest that the inhibitory effect of polyols and sugars is probably due to the complexation of transition metal ions rather than a hydroxyl radical scavenging mechanism. However, dextran, a high molecular weight polysaccharide, might function as a hydroxyl radical scavenger to protect Rix from the metal-catalyzed oxidation.

Ascorbic Acid↗

PubMed1996

Effects of reducing sugars on the chemical stability of human relaxin in the lyophilized state.

Sugars and polyols have been used routinely with lyophilized proteins and peptides as bulking agents, cryoprotectants, and lyoprotectants. However, reducing sugars may present a problem as excipients since they are potentially reactive with proteins. In this stability study of recombinant human relaxin (Rix) with various sugars as excipients in lyophilized formulations, we observed rapid covalent modifications of the protein in the presence of glucose. Analysis of the protein by LC/MS and tryptic mapping indicated two major degradation pathways. Covalent adducts of glucose with amino groups on the side chains of the protein (i.e., Lys and Arg) formed via the Maillard reaction. In addition, a significant amount of Ser cleavage from the C-terminal of the B-chain of relaxin was also identified when glucose was used as the excipient. It was observed that the latter reaction occurred to a greater extent in the solid state than in solution. We proposed a mechanism for this reaction involving an initial reaction of the Ser hydroxyl group with glucose followed by subsequent hydrolysis of the Trp-Ser amide bond via a cyclic intermediate. In contrast to glucose, mannitol (polyhydric alcohol) and trehalose (nonreducing sugar) produced stable, lyophilized formulations of Rix.

Amino Acid Sequence↗

PubMed1996

Translation initiation region plays an important role in the expression of human thrombopoietin in Escherichia coli.

A mutant human thrombopoietin (TPO) gene with a modified translation initiation region (TIR) sequence was created by site-specific mutagenesis based on the PCR technique. This mutant TPO gene encoded the same amino acid sequence as wild-type TPO gene. The wild-type TPO gene was expressed in E. coli with very low efficiency. The mutant TPO gene could reach an expression level of up to 10% of total cellular proteins in E. coli, which was much higher than the wild-type gene. The recombinant protein was mainly in the form of inclusion body which could acquire in vitro activities of human thrombopoietin after refolding.

Escherichia coli↗

PubMed1996

The mammary gland as a bioreactor: factors regulating the efficient expression of milk protein-based transgenes.

Specific regulatory regions required for hormonal regulation and tissue-specific expression of rat beta-casein and why acidic protein (WAP) genes in the mammary gland have been defined. Composite response elements with multiple binding sites for several transcription factors have been identified. Mammary gland-specific gene expression appears not to be mediated by a single transcription factor, but instead requires cooperative interactions among several factors. Signal transduction pathways regulated by lactogenic hormones result in transcription factor binding and interaction within these elements, chromatin-structure changes, and milk-protein gene expression. Intragenic sequences in the 5' and 3' untranslated regions of the beta-casein and WAP mRNAs, respectively, also appear crucial for the efficient expression of these genes. Vectors to target the expression of heterologous genes, such as insulin-like growth factor I, to the mammary gland can be designed. This technology can be used to manipulate milk composition in transgenic animals, one result being improved infant formulas.

Animals↗

PubMed1996

Nucleotide level detection of cyclobutane pyrimidine dimers using oligonucleotides and magnetic beads to facilitate labelling of DNA fragments incised at the dimers and chemical sequencing reference ladders.

We present a method for detecting cyclobutane pyrimidine dimers (CPDs) at the nucleotide level and an adaptation of Maxam-Gilbert sequencing for generating sequence reference ladders. UV irradiated genomic DNA from Escherichia coli was digested with restriction enzyme(s) and incised at the CPDs with Micrococcus luteus UV endonuclease. The subsequent specific fragments were separated using a biotin labelled oligonucleotide containing a sequence complementary to the fragments of interest and streptavidin magnetic beads. These fragments were then radiolabelled on the beads just prior to the running of the sequencing gel. For generating sequence reference ladders, the unlabelled DNA fragments of interest were base-specifically modified and subsequently cleaved at the A+G or C+T sites using the rapid Maxam-Gilbert sequencing treatments. These chemically cleaved fragments can be stored almost indefinitely. Whenever the sequence reference ladders are required, the chemically cleaved fragments can be labelled alongside the CPD-specifically incised DNA fragments using the same procedure. The adaptation of the method to detect other types of DNA damage is also discussed.

Base Sequence↗

PubMed1996

Position-dependent protein mutant profile based on mean force field calculation.

The application of the mean force field in protein mutant stability prediction is explored. Based on protein main chain characteristics, including polar fraction, accessibility and dihedral angles, the mean force field was constructed to evaluate the compatibility between an amino acid residue and its environment, from which a position-dependent protein mutant profile was constructed. At each position along a protein sequence, the native residue was replaced by the other 19 types of amino acid residues. The matches were evaluated by energies from mean force field calculation, from which a mutant profile along the protein sequence was derived. General characteristics of such a profile were analyzed. Mutant stabilities for two sets of mutants in two proteins were found to be reasonable compared with experimental data, which indicates that the present method can act as a guide in protein engineering and as an effective scoring matrix in protein sequence-structure alignment studies.

Amino Acid Sequence↗