Biomedical subjects

B Lei

Publications and source records attributed to B Lei.

At least 37 records · Page 2Linked to original sources

PubMed2000

Retinal toxicity of commercial intravitreal tissue plasminogen activator solution in cat eyes.

BACKGROUND: We previously reported retinal toxic reactions in rabbit eyes receiving intravitreal injections of commercial tissue plasminogen activator (tPA) in concentrations greater than or equal to 50 microg/0.1 mL, and recent clinical experience suggests that intravitreal tPA solution may produce toxic effects in human eyes. We therefore investigated the dose-dependent retinal toxicity of intravitreal commercial recombinant tPA solution in cat eyes, which have a vascularized inner retina and vitreous volume similar to that of human eyes. METHODS: Commercial tPA in L-arginine solution was injected into the mid vitreous cavity of normal cat eyes in doses of 25, 50, 75, and 100 microg/0.1 mL and 200 microg/0.2 mL. Control (fellow) eyes received an equal volume of sterile saline solution. After injection, eyes were evaluated by ophthalmoscopy and electroretinography for 14 days and then enucleated for histopathological evaluation. RESULTS: Fundus pigmentary alterations were observed in eyes receiving doses greater than or equal to 50 microg/0.1 mL. Changes were centered in the area around the injection site, and the area's size increased in proportion to the dosage. Mean electroretinography B-wave amplitude measured at 14 days was significantly reduced in eyes receiving greater than or equal to 50 microg of tPA in a dose-dependent fashion. Light microscopy of the involved areas showed loss of photoreceptor elements with necrosis and proliferation of the retinal pigment epithelium. CONCLUSION: Intravitreal injection of commercial tPA solution results in dose-dependent retinal toxicity in cat eyes. CLINICAL RELEVANCE: Because cat eyes are similar to human eyes regarding retinal vascularity and vitreous volume, intravitreal injections of commercial tPA (with L-arginine vehicle) in concentrations greater than 25 microg/0.1 mL are potentially unsafe in human eyes.

Animals↗

PubMed2000

Probing the mechanisms of the biological intermolecular transfer of reduced flavin.

NAD(P)H-flavin oxidoreductases [flavin reductases (FR)] are a class of enzymes capable of producing reduced flavin for bacterial bioluminescence and other biological processes. Bacterial luciferase utilizes oxygen, reduced FMN (FMNH2) and a long-chain aliphatic aldehyde as substrates for light emission. The Vibrio harveyi luciferase and FRP (for which we have cloned the gene and determined the crystal structure) is a model for the elucidation of the reduced flavin transfer mechanism using both a flavin reductase single-enzyme assay monitoring the NADPH oxidation and a flavin reductase-luciferase coupled assay measuring bioluminescence intensity or quantum output. The FRP exhibits a ping-pong kinetic pattern in the single-enzyme assay but changes to a sequential pattern in the coupled assay. Furthermore, FMN at >2x10(-6) mol/L reduced both the light intensity and quantum yield of the coupled reaction by noncompetitively inhibiting NADPH and competitively inhibiting luciferase. These results support a scheme in which the luciferase forms specific complex(es) with FRP. Indeed, such complexes were shown by fluorescence anisotropy to exist between luciferase and monomeric FRP either in the holo- or apoenzyme form. Furthermore, the reduced flavin cofactor of FRP is transferred directly to luciferase for bioluminescence, whereas the reduced flavin product of FRP is inefficient in supporting the luminescence reaction. The mechanism of reduced flavin transfer is apparently flavin and flavin reductase specific.

Bacteria↗

PubMed2000

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2000

Identification and immunogenicity of group A Streptococcus culture supernatant proteins.

Extracellular proteins made by group A Streptococcus (GAS) play critical roles in the pathogenesis of human infections caused by this bacterium. Although many extracellular GAS proteins have been identified and characterized, there has been no systematic analysis of culture supernatant proteins. Proteins present in the culture supernatant of strains of serotype M1 (MGAS 5005) and M3 (MGAS 315) mutants lacking production of the major extracellular cysteine protease were separated by two-dimensional gel electrophoresis and identified by amino-terminal amino acid sequencing and interrogation of available databases, including a serotype M1 genome sequence. In the aggregate, amino-terminal amino acid sequence data for 66 protein spots were generated, 53 unique sequences were obtained, and 44 distinct proteins were identified. Sixteen of the 44 proteins had apparent secretion signal sequences and 27 proteins did not. Eight of the 16 proteins with apparent secretion signal sequences have not been previously described for GAS. Antibodies against most of the apparently secreted proteins were present in sera from mice infected subcutaneously with MGAS 5005 or MGAS 315. Humans with documented GAS infections (pharyngitis, acute rheumatic fever, and severe invasive disease) also had serum antibodies reacting with many of the apparently secreted proteins, indicating that they were synthesized in the course of GAS-human interaction. The genes encoding four of the eight previously undescribed and apparently secreted culture supernatant proteins were cloned, and the proteins were overexpressed in Escherichia coli. Western blot analysis with these recombinant proteins and sera from GAS-infected mice and humans confirmed the immunogenicity of these proteins. Taken together, the data provide new information about the molecular aspects of GAS-host interactions.

Amino Acid Sequence↗

PubMed2000

Human melanoma-associated retinopathy (MAR) antibodies alter the retinal ON-response of the monkey ERG in vivo.

PURPOSE: Melanoma-associated retinopathy (MAR) is a paraneoplastic condition that causes visual symptoms of night-blindness and photopsias. The electroretinogram (ERG) of MAR patients is characteristically abnormal in a way that implicates retinal depolarizing bipolar cell (DBC) dysfunction. Whether an injection of IgG from MAR patients into the vitreous of monkeys would alter the ERG acutely as a demonstration of a functional basis for patients' visual symptoms was explored. METHODS: MAR IgG was isolated from three visually symptomatic melanoma patients. Control IgG was from melanoma patients with no vision problems. The ERG was monitored after intravitreal injections into monkey eyes. One eye was injected with 2-amino-4-phosphonobutyric acid (APB), which is known to block DBC ON-pathway responses. Retinal immunocytochemistry was performed using fluorescein isothiocyanate-labeled goat anti-human IgG. RESULTS: Within 1 to 3 hours after MAR IgG injection, the ERG photopic b-wave was diminished, with far less effect on the a- and d-waves. These changes are characteristic of DBC dysfunction and were similar to the effects of APB. The scotopic ERG b-wave, which reflects activity of rod-driven DBCs, showed a loss of amplitude and threshold sensitivity after MAR IgG. Retinal immunocytochemistry with anti-IgG antibody showed IgG penetration throughout the retinal layers, but staining was not specific for a single type of retinal neuron. CONCLUSIONS: Intravitreal injection of human MAR IgG altered the monkey ERG acutely in ways that implicate functional disruption of retinal DBC signaling. These results support the hypothesis that MAR IgG circulating antibodies are responsible for the reported visual symptoms. Bipolar cells in the ON-pathway appear to be affected more than OFF-pathway bipolar cells of the cone pathway in this acute preparation.

Aminobutyrates↗

PubMed2000

[Effect of long-term no-tillage and application of organic manure on some properties of soil fertility in rice/wheat rotation].

A field experiment from June 1983 to October 1997 was conducted to study the effect of long-term no-tillage and application of manure on soil fertility properties. The results showed that the contents of soil organic C, total N and available N in the treatment of fourteen consecutive year no-tillage were all significantly higher in 0-5 cm soil layer, while, lower in 5-10 and 10-20 cm layers than in conventional tillage. The content of water stable aggregate in 0-20 cm layer was increased in no-tillage in that the soil structure was less destroyed. Based on various soil fertility properties, the contribution order of different fertilization treatments to soil fertility was as follows: pig manure > straw > green manure > chemical fertilizer > no fertilizer.

Manure↗

PubMed2000

[Study on uptake of fluoroquinolones by Staphylococcus aureus].

This study aimed to investigate the mechanism of Staphylococcus aureus' resistance to fluoroquinolones. The uptake of lomefloxacin, ofloxacin by Staphylococcus aureus and the effect of CCCP on the uptake were studied by the fluorescence method. The uptake of lomefloxacin, ofloxacin by sensitive organisms is higher than that by resistant organisms; CCCP had different influences on different organisms, but no significant difference between the effects of CCCP on sensitive strains and on resistant strains was noted. The uptake of lomefloxacin, ofloxacin by SA2-16 was much lower than that by SA2-16's parent, and after the addition of CCCP, the uptake of both quinolones by SA2-16 increased rapidly, but the concentration did not reach its parent's concentration in stable condition. These results indicate that the decreasing of the uptake of fluoroquinolones is one of the mechanisms responsible for Staphylococcus aureus to resistance fluoroquinolones, and as a mechanism of resistance to drugs, it plays different roles in different strains.

Anti-Infective Agents↗

PubMed1999

Relationship between the conserved alpha subunit arginine 107 and effects of phosphate on the activity and stability of Vibrio harveyi luciferase.

The Arg107 of the alpha subunit is a conserved residue for all known bacterial luciferases. The phosphate moiety of the reduced flavin mononucleotide (FMNH(2)) side chain has been hypothesized to be anchored at this site (A. J. Fisher, F. M. Raushel, T. O. Baldwin, and I. Rayment Biochemistry 34, 6581-6586, 1995). Mutations of alphaArg107 of the Vibrio harveyi luciferase to alanine, serine, and glutamate were carried out to test such a hypothesis. These variants were characterized and compared with the wild-type luciferase with respect to their K(m) for decanal, FMNH(2), and reduced riboflavin in both low- (0.01 or 0.05 M) and high- (0.3 M) phosphate buffers at pH 7.0. Results are consistent with the hypothesized binding of the FMNH(2) phosphate group by alphaArg107. Moreover, the alphaArg107 residue was apparently important in the expression of the luciferase maximal activity and aldehyde binding. Phosphate ion is also known to have other effects on luciferase stability. We compared the three luciferase variants with the native enzyme with respect to the decay rate of the FMN 4a-hydroperoxide intermediate II, and rates of inactivation by trypsin digestion, modification by N-ethylmaleimide, and heat treatment in low- and high-phosphate buffers. On the basis of patterns of the phosphate effects, alphaArg107 appeared to be important to the enhancement of luciferase stability against trypsin proteolysis at high phosphate but was not involved in regulating the intermediate II decay or sensitivity to N-ethylmaleimide modification. Differential effects of mutations on luciferase thermal stability were observed. It is uncertain whether alphaArg107 is involved in the enhanced thermal stability of the native luciferase in high phosphate buffer.

Alanine↗

PubMed1999

Sepsis facilitates brain serotonin activity and impairs learning ability in rats.

Sepsis often provokes various neurological disorders. Because many neurologic symptoms are caused by changes in neurotransmissions, we investigated the relationship between behavioral alterations and changes in activities of the monoaminergic systems in rats. Sepsis was induced by cecal ligation and puncture. A step-through passive avoidance test was used for the behavioral evaluation. Passive avoidance retention in animals subjected to learning immediately before the septic or sham operation was examined after 24 or 48 h. Retention performance in animals subjected to learning 24 h after the operation was also examined after a further 24 h. Plasma concentrations of amino acids were determined 24 h after the operation. The activities of the brain monoaminergic systems were evaluated by ratios of metabolites to monoamines. Marked damage was found in the septic rats in the blood analysis 24 h after the operation. The plasma concentrations of tyrosine and arginine in the septic rats were decreased to 69% and 70% of those in the sham-operated animals, respectively. Retention performance was impaired in the septic rats when they were subjected to learning 24 h after the operation, but it was not impaired when animals were subjected to learning before the septic operation. The brain concentration of serotonin was increased in the cerebral cortex, striatum, and hippocampus 48 h after the septic operation, but not after 24 h. The concentration of 5-hydroxyindoleacetic acid, a metabolite of serotonin, was increased in the above three regions both 24 and 48 h after the operation, but not in the hypothalamus. Facilitation of the serotonergic activity in the telencephalon and hippocampus is suggested to be involved in the impairment of learning ability in sepsis.

Animals↗

PubMed1999

Measurement of total nitric oxide metabolite (NO(x)(-)) levels in vivo.

The measurement of the total level of nitric oxide (NO) metabolite (NO(x)(-)) by microdialysis has recently been used to assess the production of NO in the in vivo brain [D. Luo, S. Knezevich, S.R. Vincent, N-Methyl-D-aspartate-induced nitric oxide release: an in vivo microdialysis study, Neuroscience, 57 (1993), 897-900; K. Ohta, N. Arai, M. Shibata, J. Hamada, S. Komatsumoto, K. Shimazu, Y. Fukuuchi, A novel in vivo system for consecutive measurement of brain nitric oxide production combined with the microdialysis technique, Neurosci. Lett., 176 (1994), 165-168; K. Shintani, S. Kanba, T. Nakai, K. Sato, G. Yagi, R. Kato, M. Arai, Measurement by in vivo microdialysis of nitric oxide release in the rat cerebellum, J. Psychiatr. Neurosci., 3 (1994), 217-221; H. Togashi, K. Mori, K. Ueno, M. Matsumoto, N. Suda, H. Saito, M. Yoshika, Consecutive evaluation of nitric oxide production after transient cerebral ischemia in the rat hippocampus using in vivo brain microdialysis, Neurosci. Lett., 240 (1998), 53-57]. Although several methods are available for detecting NO(x)(-) levels in dialysates, these methods are either not sensitive enough or require expensive experimental equipment. The method described herein provides a convenient and sensitive procedure for determining NO(x)(-) levels in dialysates. This method is useful for the in vivo study of NO production from various brain regions in various pathological conditions, and can be applied to other tissues.

Animals↗

PubMed1999

Nitric oxide production in the CA1 field of the gerbil hippocampus after transient forebrain ischemia : effects of 7-nitroindazole and NG-nitro-L-arginine methyl ester.

BACKGROUND AND PURPOSE: The present study was designed to examine the time course of nitric oxide (NO) production and the source of NO in the CA1 field of the gerbil hippocampus after transient forebrain ischemia. METHODS: The production of NO in the CA1 field of the hippocampus after transient ischemia was monitored consecutively by measuring total NO metabolites (NOx-, NO2- plus NO3-) with the use of brain microdialysis. 7-Nitroindazole (7-NI) and NG-nitro-L-arginine methyl ester were used to dissect the relative contributions of neuronal NO synthase and endothelial NO synthase to the NO production. The histological outcomes of 7-NI in 5- and 10-minute global ischemia were also evaluated. RESULTS: The production of NO in the CA1 field of the hippocampus after ischemia was dependent on the severity of ischemia. Ischemia for 2 or 5 minutes did not induce a significant increase in NOx- levels in the CA1 field of the hippocampus after reperfusion, whereas the 10- and 15-minute ischemias produced significant and persistent increases in NOx- levels. 7-NI did not inhibit the basal NOx- levels and showed no effects on NOx- levels after 5 minutes of ischemia. However, it completely inhibited the increased NOx- levels after 10 or 15 minutes of ischemia. 7-NI provided minor neuroprotection in 5 minutes but not in 10 minutes of global ischemia. CONCLUSIONS: The increased NO level in the CA1 field of the hippocampus after ischemia is produced mostly by neuronal NO synthase, whereas the basal NO level mainly originates from endothelial NO synthase. The observed neuroprotective effect of 7-NI in 5-minute global ischemia in gerbils may not be due to neuronal NO synthase inhibition by this drug.

Animals↗

PubMed1999

[Evaluation of effectiveness of hepatitis B plasma derived vaccine and a survey of hepatitis C virus infection in preschool children in Chengdu, Sichuan].

This study was undertaken to evaluate the effectiveness of hepatitis B plasma-derived vaccine and investigate the character of hepatitis C virus infection in preschool children. The anti-HBs, HBsAg and anti-HCV of 985 plasma samples were examined by ELISA. The results showed that the positive rate of HBsAg in the children (aged < 7 yr) in the communities was 0.89% and the effectiveness of the vaccine was 91.38%. The positive rate of anti-HBs decreased with the increase in age; the positive rate of HBsAg did not increase with age. The positive rate of HBsAg was as high as 4.69% in the children from hospitals, so supervision and enhanced vaccination should be administered properly in this group. The positiverate of anti-HCV was 1.18% in the communities, but 3.17% in hospitals. It is worthy to investigate how children get infected with HCV.

Child, Preschool↗

PubMed1999

[A study on porins of Escherichia coli HX88108].

To investigate the relationship between porins deficiency and the resistance of Escherichia coli HX88108. The porins of Escherichia coli HX88108 were studied by SDS-PAGE and were compared with the porins of standard strains. The result showed that E. coli HX88108 did not lose OmpF and OmpC, and the molecular weights of these two porins were 38 kd and 37 kd respectively. There is no relationship between the resistance of Escherichia coli HX88108 and porins deficiency.

Drug Resistance, Bacterial↗

PubMed1999

[Sub-cloning and preliminary sequence analysis of the gene encoding of a cefoperazone hydrolyzing beta-lactamase isolated from Escherichia coli].

Escherichia coli HX88108, which is resistant to cefoperazone(CPZ), was isolated from a severely infected patient. We studied genetical basis of beta-lactamase produced in E. coli HX88108 by pFL25, one of the recombinant plasmid of pFC. Largescale pFL25 plasmid was extracted, purified, and cleaved with restriction endonuclease EcoR I, Sal I, Pvu I, then subcloned into vector pUC19 as 1.9 kb, 0.9 kb, 0.65 kb fragments respectively. Recomminant plasmids were selected by alpha-complementation and determined by restriction endonuclease analysis. DNA sequencing was performed by the dideoxy polymerase chain termination method. Partial nucleotide sequence(1-78 nucleotide position) of the gene was found to be highly homologous (97%) with the gene coding for TEM-52 extended spectrum beta-lactamase of K. pneumonise, suggesting that the beta-lactamase coded by the cefoperazone resistant gene might be derived from TEM-type beta-lactamase.

Anti-Bacterial Agents↗

PubMed1998

Mechanism of reduced flavin transfer from Vibrio harveyi NADPH-FMN oxidoreductase to luciferase.

The mechanisms of reduced flavin transfer in biological systems are poorly understood at the present. The Vibrio harveyi NADPH-FMN oxidoreductase (FRP) and the luciferase pair were chosen as a model for the delineation of the reduced flavin transfer mechanism. FRP, which uses FMN as a cofactor to mediate the reduction of the flavin substrate by NADPH, exhibited a ping-pong kinetic pattern with a Km, FMN of 8 microM and a Km,NADPH of 20 microM in a single-enzyme spectrophotometric assay monitoring the NADPH oxidation. However, the kinetic mechanism of FRP was changed to a sequential pattern with a Km,FMN of 0.3 microM and a Km,NADPH of 0.02 microM in a luciferase-coupled assay measuring light emission. In contrast, the Photobacterium fischeri NAD(P)H-FMN oxidoreductase FRG showed the same ping-pong mechanism in both the single-enzyme spectrophotometric and the luciferase-coupled assays. Moreover, for the FRP, FMN at concentrations over 2 microM significantly inhibited the coupled reaction in both light intensity and quantum yield, and showed apparent noncompetitive and competitive inhibition patterns against NADPH and luciferase, respectively. No inhibition of the NADPH oxidation was detected under identical conditions. These results are consistent with a scheme that the reduced flavin cofactor of FRP is preferentially utilized by luciferase for light emission, the reduced flavin product generated by the reductase is primarily channeled into a dark oxidation, and luciferase competes against flavin substrate in reacting with the FRP reduced flavin cofactor. An FRP derivative containing 2-thioFMN as the cofactor was also used to further examine the mechanism of flavin transfer. Results again indicate a preferential utilization of the reductase reduced flavin cofactor by luciferase for the bioluminescence reaction.

Apoenzymes↗

PubMed1998

Measurement of the extracellular H2O2 in the brain by microdialysis.

This paper reports on the protocol for the determination of H2O2 in the brain using in vivo microdialysis coupled with fluorometry of dichlorofluorescin oxidation. We applied this protocol to monitor changes in the concentration of H2O2 in the brain, in vivo, during ischemia and reperfusion. Using this method, changes in the level of H2O2 in the brain during ischemia and reperfusion were effectively determined. The present protocol provides a novel tool to study the production of reactive oxygen species in the brain.

Animals↗

PubMed1998

[Influence of moderate hypothermia on the contents of 6-KETO-PGF1 alpha and TXB2 in brain tissues after cardiac arrest and resuscitation in dogs].

To investigate the effect of moderate hypothermia of whole brain (30 degrees C to 32 degrees C) on the levels of prostacyclin and thromboxane A2 in brain tissues after cardiac arrest and resuscitation. Twenty-one dogs were divided into four groups: Group A, nonischemic controls (n = 4); Group B, 15 minute cardiac arrest without reperfusion (n = 4); Group C, 15 minute cardiac arrest and standard resuscitation (n = 6); Group D, 15 minute cardiac arrest and moderate hypothermia (30 degrees C-32 degrees C). The results showed that as compared with those in Group A, the 6-Keto-PGF1 alpha levels remained substantially unchanged in Group C and D (P > 0.05), and the levels of TXB2 and TXB2/6-Keto-PGF1 alpha ratio increased significantly in Group C (P < 0.01), but both maintained basically stable in Group D (P > 0.05). In comparison with those in Group C, the levels of TXB2 and TXB2/6-Keto-PGF1 alpha ratio decreased dramatically (P < 0.01). It is concluded that the moderate hypothermia can depress the arachidonic acid metabolism and keep a certain balance between the activities of TXA2 and PGI2 during cerebral resuscitation.

6-Ketoprostaglandin F1 alpha↗

PubMed1997

The effect of dopamine depletion on the H2O2 production in the rat striatum following transient middle cerebral artery occlusion.

The changes in the extracellular concentrations of rat striatal H2O2, dopamine (DA) and its metabolites during middle cerebral artery (MCA) occlusion and reperfusion were simultaneously examined by microdialysis, and the relationship between the ischemia-induced release of DA and the generation of H2O2 was estimated by assessing the effect of the lesion of the substantia nigra (SN). In the rats without SN lesions, a significant increase in the striatal H2O2 level was observed during the ischemia and reperfusion phases. In the rats with SN lesions, the ischemia-induced H2O2 production was not attenuated. These results suggest that DA is not an important source of H2O2 in cerebral ischemia and reperfusion.

3,4-Dihydroxyphenylacetic Acid↗