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Biomedical subjects

L Yu

Publications and source records attributed to L Yu.

At least 415 records · Page 23Linked to original sources
PubMed1997

Resolution and reconstitution of succinate-ubiquinone reductase from Escherichia coli.

A modified procedure is developed for isolation of highly purified succinate-ubiquinone reductase from Escherichia coli NM256 containing a cloned sdh operon in a multicopy plasmid. Succinate-ubiquinone reductase is solubilized from the membrane by polyoxyethylene-9-lauryl ether and purified by DEAE-Sepharose CL-6B column chromatography. The isolated reductase is resolved into a reconstitutively active, two-subunit succinate dehydrogenase and a two-subunit membrane anchoring protein fraction (the SdhC-SdhD fraction) by alkaline (pH 10.2) treatment of the reductase in the presence of 1 M urea, followed by DEAE-Sepharose CL-6B column chromatography under anaerobic conditions. Isolated succinate dehydrogenase and the SdhC-SdhD fraction alone show no succinate-ubiquinone reductase activity. However, when a given amount of the SdhC-SdhD fraction is mixed with varying amounts of succinate dehydrogenase or vice versa succinate-ubiquinone reductase activity increases as the amount of succinate dehydrogenase or the SdhC-SdhD fraction added increases. Maximum reconstitution is obtained when the weight ratio of succinate dehydrogenase to the SdhC-SdhD fraction reaches 5.26. This ratio is slightly higher than the calculated value of 3.37, obtained by assuming 1 mol of succinate dehydrogenase reacts with 1 mol of SdhC and SdhD. The isolated SdhC-SdhD fraction contains 35 nmol cytochrome b556/mg protein. Unlike mitochondrial cytochrome b560, the cytochrome b556 is reducible by succinate in the isolated and complex forms. Furthermore, cytochrome b556 in the isolated SdhC-SdhD fraction has absorption properties, carbon monoxide reactivity, and EPR characteristics similar to those of cytochrome b556 in intact succinate-ubiquinone reductase, indicating that its heme environments are not affected by the presence of succinate dehydrogenase. However, the redox potential of cytochrome b556 in the SdhC-SdhD fraction (22 mV) increases slightly when complexed with succinate dehydrogenase (34 mV). No hybrid succinate-ubiquinone reductase is formed from mitochondrial QPs (the membrane-anchoring protein fraction of bovine heart mitochondrial succinate-ubiquinone reductase) and E. coli succinate dehydrogenase or vice versa. However, the cytochrome b556 in E. coli SdhC-SdhD fraction is reducible by succinate in the presence of mitochondrial succinate dehydrogenase, and the rate of cytochrome b556 reduction correlates with the reconstitutive activity of the mitochondrial succinate dehydrogenase.

Animals↗
PubMed1997

Conversion of desulforedoxin into a rubredoxin center.

Rubredoxin and desulforedoxin both contain an Fe(S-Cys)4 center. However, the spectroscopic properties of the center in desulforedoxin differ from rubredoxin. These differences arise from a distortion of the metal site hypothesized to result from adjacent cysteine residues in the primary sequence of desulforedoxin. Two desulforedoxin mutants were generated in which either a G or P-V were inserted between adjacent cysteines. Both mutants exhibited optical spectra with maxima at 278, 345, 380, 480, and 560 nm while the low temperature X-band EPR spectra indicated highspin Fe3+ ions with large rhombic distortions (E/D = 0.21-0.23). These spectroscopic properties are distinct from wild type desulforedoxin and virtually identical to rubredoxin.

Cysteine↗
PubMed1997

Effects of maternal oral administration of monosodium glutamate at a late stage of pregnancy on developing mouse fetal brain.

Monosodium glutamate (MSG) was shown to penetrate placental barrier and to distribute to embryonic tissues using [3H]glutamic acid ([3H]Glu) as a tracer. However, the distribution is not even; the uptake of MSG in the fetal brain was twice as great as that in the maternal brain in Kunming mice. Other maternal mice were given per os MSG (2.5 mg/g or 4.0 mg/g body weight) at 17-21 days of pregnancy, and their offspring behaviors studied. The results showed that maternal oral administration of MSG at a late stage of pregnancy decreased the threshold of convulsion in the litters at 10 days of age. Y-maze discrimination learning was significantly impaired in the 60-day-old filial mice. On the other hand, no significant difference in spatial learning or tail flick latency was measured between the experimental animals and the controls. The filial mice of MSG-treated mothers could either not grasp a rope tightly, or grasped the rope tightly but could not crawl along the rope at the beginning of the training. However, such mice, after training, could grasp and crawl along the rope as well as controls. Obvious neuronal damage was not detected in the periventricular organs or the hypothalamus under a light microscope. The rate of weight gain for experimental animals was greater than for controls throughout the period from 20 to 90 days. Mating of treated males with treated females resulted in pregnancies and normal offspring, indicating that oral administration of MSG at a late stage of pregnancy did not affected the reproductive capacity of the offspring. The possible differences and relationship between MSG-induced damage to developing human and rodent brain are discussed.

Administration, Oral↗
PubMed1997

Assessing linkage on chromosome 5 using components of variance approach: univariate versus multivariate.

We report results when one disease related quantitative trait (Q1) is analyzed versus multiple related quantitative traits using a components of variance approach [Amos, 1994; de Andrade and Amos, 1996]. In both cases, we used age, sex and environmental factor (EF) as covariates. Analysis with ascertainment correction of samples selected through probands with extremely high trait values was also performed and is presented. Testing procedures to detect linkage using the components of variance approach are discussed. The univariate and multivariate analyses showed effects of locus 15 on chromosome 5 only for Q1. This result was confirmed when using ascertainment correction.

Analysis of Variance↗
PubMed1997

Enhancement of cartilage matrix protein synthesis in arthritic cartilage.

OBJECTIVE: To investigate whether the synthesis of cartilage matrix protein (CMP) is enhanced in arthritic cartilage. METHODS: The content of CMP in human and pig cartilage was determined by immunoblotting, and CMP-producing chondrocytes in osteoarthritic (OA) and rheumatoid arthritic (RA) joints were immunostained. RESULTS: CMP was undetectable in the condylar cartilage and disc of pigs, whereas it was abundant in the rib and tracheal cartilage of the same animals. By immunohistochemical analysis, CMP was localized in only a few chondrocytes (5%) in normal human joints, whereas numerous chondrocytes (>60%) were immunostained in RA joints. The number of CMP-producing cells was also increased in OA cartilage (>40%). Immunoblotting analyses confirmed that the CMP content in the cartilage from OA and RA patients was much higher than that in normal cartilage. CONCLUSION: These findings demonstrate that articular chondrocytes can synthesize CMP, although it is suppressed under physiologic conditions. The results also suggest that articular chondrocytes express CMP in response to arthritic stimuli.

Adult↗
PubMed1997

Atomic force microscopy as a tool for study of human hair.

Atomic force microscopy (AFM) is a newly developed microscopic technique that offers high-resolution power, less intrusive measurement, and requires little sample pretreatment for elucidating structures of biological materials in three dimensions and in their natural environment. In this study, AFM has been used not only as an imaging technique for examining human hair structure at high resolution, but also as a tool for quantitative assessment of the effect of treatment in 10 mM phosphate buffered saline of pHs 3.0, 7.0, and 11.0 and heating on human hair structure. It is observed that the hair cuticle is a sensitive indicator of external influences on hair structure, and that its height can be used as a parameter for quantitative assessment. The experimental results obtained show that the swelling of hair caused by the incubation in the buffer decreases with the increase of the pH values and that, depending on the duration of heating, the hair undergoes structural expansion and shrinkage. This study demonstrates that AFM can be used as a valuable alternative to conventional microscopic techniques for hair research.

Asian↗
PubMed1997

Helper virus-free herpes simplex virus-1 plasmid vectors for gene therapy of Parkinson's disease and other neurological disorders.

Vectors based on herpes simplex virus type 1 (HSV-1) have potential for gene therapy of neurological disorders. HSV-1 plasmid vectors (amplicons) contain only approximately 1% of the 150-kb HSV-1 genome and have been packaged into virus particles by using a helper virus. We have demonstrated that HSV-1 plasmid vectors which express tyrosine hydroxylase can cause long-term biochemical and behavioral recovery in the 6-hydroxydopamine rat model of Parkinson's disease. Furthermore, we and others have used HSV-1 plasmid vectors which express a wide range of genes that affect neuronal physiology. Because of the pathogenicity of the HSV-1 helper virus, however, the use of this vector system has been limited to studies in animal models or primary cultures of neural cells. Thus, to increase the safety of HSV-1 plasmid vectors, we recently developed a helper virus-free packaging system that may facilitate studies on neuronal physiology and potential therapeutic applications.

Animals↗
PubMed1997

T cell receptor gene polymorphisms associated with anti-insulin, autoimmune T cells in diabetes-prone NOD mice.

The great majority of insulin-specific T cell clones isolated from islets of NOD mice react with insulin peptide B-(9-23) (amino acids 9-23 of the insulin B chain). The T cell receptors of these clones contain diverse beta-chains but restricted alpha-chains. The dominant alpha-chain motif is a V alpha 13 segment (10 out of 13) combined to either a J alpha 45 or a J alpha 34 segment (eight out of 13). Furthermore, nine out of 10 of these V alpha 13 segments are a product of a novel NOD TCR V alpha gene which we have termed V alpha 13.3. Analysis of V alpha 13 transcripts from splenic cDNA libraries from the NOD, BALB/c and C57BL/6 mice revealed significant differences between strains. The NOD sequences contained both V alpha 13.1 and the novel V alpha 13.3. The BALB/c contained the previously reported V alpha 13.1 and V alpha 13.2, but not the V alpha 13.3 sequence identified in the NOD anti-insulin T cell clones. The C57BL/6 had V alpha 13.1 and V alpha 13.3 plus two additional novel sequences which we have termed V alpha 13.4 and V alpha 13.5. These V alpha 13 subfamily members differed by two to four amino acids in either the CDR1 region or adjoining the CDR2 region. The frequency of utilization of the different V alpha 13 subtypes varied dramatically between strains. In the NOD spleen, V alpha 13.3 was detected 79% of the time, compared to 21% for V alpha 13.1. In contrast, the C57BL/6 spleen contained only 7% of V alpha 13.3 sequences compared to the other subfamily members present (V alpha 13.1: 27%; V alpha 13.4: 56%; V alpha 13.5: 10%). MHC polymorphisms or other unknown selective pressures may contribute to these differences in V alpha 13 utilization. We hypothesize that the presence and frequent utilization of the V alpha 13.3 T cell receptor element is involved in targeting insulin B-(9-23) and may be related to diabetes susceptibility of NOD mice.

Animals↗
PubMed1997

T-cell reactivity to GAD65 peptide sequences shared with coxsackie virus protein in recent-onset IDDM, post-onset IDDM patients and control subjects.

GAD65 is one of the major autoantigens associated with insulin-dependent diabetes mellitus (IDDM). The two peptides p17 and p18 of GAD65 that share sequence similarity with coxsackie virus (amino acid sequence identity: PEVKEK) appeared to be the major determinants of GAD65 recognized preferably by T cells from new-onset IDDM patients and their first degree relatives. In contrast, in our study unrelated control subjects frequently recognized the two GAD peptides (55%, 16/29), similar to first degree relatives (41%, 12/29) and IDDM patients post-onset (68%, 15/22). However, recent-onset IDDM patients, responded less frequently (25%, 4/16) compared with IDDM patients post-onset (p < 0.03) or unrelated control subjects (borderline significant) confirming previous observations in humans and NOD mice that T-cell reactivity to GADp17/p18 at diabetes onset is decreased. Moreover, this study demonstrated a positive correlation of T-cell proliferation to GAD p17 (amino acid 247-266) and p18 (amino acid 260-279) with simultaneous responses to both peptides in 13% of all subjects tested (n = 97) (p < 0.001). T-cell proliferation to GAD p17 was higher than to p18 in recent-onset diabetic patients, first degree relatives and unrelated control subjects (p < 0.02, p < 0.004, p < 0.002, respectively). However, in post-onset IDDM patients, the two peptides were recognized equally well. Our results show that T-cell reactivity to GAD65, peptides homologous with coxsackie protein is very frequently observed, but not primarily associated with IDDM. The temporary decline of T-cell proliferation is not associated with the beta-cell destruction process, but with clinical manifestation. The positive correlation of reactivity to the two peptides in the viral motif implicates that PEVKEK is an immunogenic epitope.

Adolescent↗
PubMed1997

Altered immune response to insulin in newly diagnosed compared to insulin-treated diabetic patients and healthy control subjects.

Insulin-dependent diabetes mellitus (IDDM) is the result of a T-cell mediated autoimmune beta-cell destruction, which is accompanied by autoantibodies. We analysed the cellular and humoral immune response to insulin and insulin peptides in patients with recent-onset IDDM, IDDM patients treated with insulin, non-diabetic first degree relatives and unrelated control subjects. There were no differences in T-cell reactivity to whole insulin or insulin peptides in general between age-matched groups of IDDM patients, relatives or healthy control subjects. In contrast to investigations in NOD mice, no immunodominant or disease-specific insulin peptide could be identified. Surprisingly, a positive correlation of T-cell responses to insulin with age was noticed (p < 0.005). This resulted in an inverse relation of insulin autoantibodies (IAA) and insulin reactive T-cells (p < 0.001) together with the well-described negative correlation of IAA with age. Interestingly, insulin-treated patients differed from age-matched recent-onset IDDM patients: first, simultaneous immune recognition of insulin with T-cells and IAA was only seen in patients treated for 6 months with insulin; second, insulin-treated patients rarely responded to whole insulin; third, they displayed less determinant spreading, and finally, recognition of multiple insulin peptides was not accompanied by crossreactivity to whole insulin. These distinct observations in insulin-treated IDDM patients, together with the inverse correlation between humoral and cellular responses to insulin, may result from activation or modulation of different T-cell subsets, and may be of relevance to insulin therapy trials, in which selective activation of non-destructive T-cell subsets may be a key to successful intervention.

Adolescent↗
PubMed1997

Investigation of the image contrast of tapping-mode atomic force microscopy using protein-modified cantilever tips.

In this work we have designed a simple system to investigate empirically the image contrast of tapping-mode atomic force microscopy (TMAFM). We modified the cantilever tips with protein molecules (bovine serum albumin or goat anti-biotin antibody) and used these protein-modified cantilevers to scan poly-L-lysine films and antibody layers deposited on mica in air under ambient conditions. We also investigated the effects of manipulating the setpoint voltage in this system. It was found that extra topographic features with a patchlike appearance were introduced into the TMAFM images of both the poly-L-lysine and antibody films when scanned with the protein-modified tips, even at initial preset setpoints, and were superimposed on the topography of the samples. The surface coverage of the patchlike features in the TMAFM images changes significantly with the setpoint voltage in a reversible and nonlinear manner. These are believed to arise from the surface indentation of the sample or from the structural deformation of the proteins at the tip induced in TMAFM imaging. Interestingly, it was observed in the experiment that no structural alteration or damage was discernible on the sample surface, even after continuous scanning with the protein-modified tips for a long period of time, with varying setpoint voltage. This study provides experimental evidence that cantilever tips modified with protein molecules or, under certain circumstances, even unmodified tips introduce extra topographical features (i.e., artifacts) and enhance the image contrast of TMAFM imaging of soft materials, which is dependent on their mechanical properties.

Aluminum Silicates↗
PubMed1997

Proposal of invasiveness score to predict recurrence and survival after curative hepatic resection for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most malignant human tumors and is associated with a high incidence of postoperative recurrence. There is no generally accepted definition for HCC invasiveness. Moreover, the predictive value of the pathologic factors that reflect HCC invasiveness was previously studied as separate events, with much controversy among different study groups. In this study, we proposed an invasiveness scoring system based on the relative importance of six criteria for HCC invasiveness: portal vein invasion, intrahepatic metastasis, hepatic vein invasion, serosal invasion, absence of tumor capsule, or presence of capsular invasion. METHODS: A total of 137 patients (111 male and 26 female) who underwent curative hepatectomy for HCC were included. Scoring of the six pathologic parameters was based on the clinical significance of each parameter as a single predictor for recurrence after curative resection. According to our scoring system, the patients were divided into three groups: low invasive HCC group A with a total invasiveness score 0 to 1, moderately invasive group B with a score of 2 to 4, and highly invasive group C with a total score of 5 or greater (5 to 11 points). RESULTS: Evaluation of the current scoring system showed a significant stepwise increase in the incidence of recurrence as the invasiveness score increased. Moreover, disease-free survival was significantly different among the three groups (log rank p < 0.0001). The 1-, 3-, 5-, and 8-year disease-free survival rates were 89%, 59%, 54%, and 54% in group A; 72%, 32%, 12%, and 10% in group B; and 54%, 19%, 7%, and 0 in group C, respectively. Multivariate analysis showed that the patients of groups B and C had a significantly worse prognosis compared with those of group A (p < 0.0001). CONCLUSIONS: The current scoring system can classify HCCs into three invasive categories and predict more accurately recurrence and disease-free survival after curative hepatectomy compared with any single invasive parameter previously proposed. Moreover, this system can be used as a therapeutic guide during and after the surgical decision making.

Adult↗
PubMed1997

Epstein-Barr virus BHRF1 prohibits the cells of nasopharyngeal carcinoma from apoptosis.

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). The BHRF1 EBV protein is expressed at high levels in productively infected cells and certain latently infected cells. In order to investigate the effect of expression of BHRF1 on the biological behaviour of NPC cells, we constructed the BHRF1 high expression vector and transfected it into the NPC cell line, CNE2. Then, the alteration of proliferation and apoptotic rates in the cells were tested before and after camptothecin treatment. After treatment by camptothecin, BHRF1-CNE2 cells could constantly and slowly proliferate and its apoptotic rate was less than in control groups, and the number of cells in the G phase decreased and in the S phase increased. So, it suggests that BHRF1 expression can enhance the resistibility of CNE2 cells to DNA-damaging agents that cause apoptosis.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Solution structure of an rRNA methyltransferase (ErmAM) that confers macrolide-lincosamide-streptogramin antibiotic resistance.

The Erm family of methyltransferases is responsible for the development of resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics. These enzymes methylate an adenine of 23S ribosomal RNA that prevents the MLS antibiotics from binding to the ribosome and exhibiting their antibacterial activity. Here we describe the three-dimensional structure of an Erm family member, ErmAM, as determined by NMR spectroscopy. The catalytic domain of ErmAM is structurally similar to that found in other methyltransferases and consists of a seven-stranded beta-sheet flanked by alpha-helices and a small two-stranded beta-sheet. In contrast to the catalytic domain, the substrate binding domain is different from other methyltransferases and adopts a novel fold that consists of four alpha-helices.

Amino Acid Sequence↗
PubMed1997

Identification of the polymorphically expressed CYP2C19 and the wild-type CYP2C9-ILE359 allele as low-Km catalysts of cyclophosphamide and ifosfamide activation.

Cyclophosphamide and ifosfamide are alkylating agent prodrugs that require activation by cytochrome P450 (CYP) to manifest their cancer chemotherapeutic activity. The present study investigates the activity of four individual human CYP2C enzymes and their allelic variants in cyclophosphamide and ifosfamide activation as an initial attempt to gain insight into the underlying basis for the large interpatient differences in the clinical pharmacokinetics and metabolism of these anticancer drugs. Recombinant CYP2C8, CYP2C19, two allelic variants of CYP2C18, and six variants of CYP2C9 expressed in a yeast cDNA expression system were each enzymatically active, as judged by the ability of the isolated microsomes to catalyse 7-ethoxycoumarin O-deethylation after reconstitution with purified NADPH-cytochrome P450 reductase and cytochrome b5. With cyclophosphamide as substrate, CYP2C19 had the lowest apparent Km, followed by CYP2C9, CYP2C18 and CYP2C8, whereas in the case of ifosfamide, the rank order was: Km CYP2C19 < CYP2C18 < CYP2C9 < CYP2C8. CYP2C18 had the highest in vitro intrinsic clearance/catalytic efficiency (apparent Vmax/Km) in cyclophosphamide and ifosfamide activation, followed by 2C19 > 2C9 approximately 2C8. Examination of a panel of CYP2C allelic variants revealed that CYP2C18-Thr385 had both a higher Vmax and a higher apparent Km toward cyclophosphamide than CYP2C18-Met385 with no difference in catalytic efficiency, whereas with ifosfamide the Thr385 allele exhibited a strikingly lower apparent Km resulting in a six-fold higher catalytic efficiency. In the case of CYP2C9, a Ile359 to Leu mutation associated with poor metabolism of the hypoglycemic drug tolbutamide decreased catalytic efficiency toward cyclophosphamide by increasing the apparent Km, whereas the same mutation reduced the efficiency of this P450 toward ifosfamide by decreasing the Vmax. Substitution of CYP2C9-Gly417 by Asp resulted in a two-fold lower catalytic efficiency for cyclophosphamide metabolism but a three-fold higher efficiency for ifosfamide metabolism. A His276 to Gly substitution resulted in an increase in both Vmax and apparent Km with no net change in catalytic efficiency for either oxazaphosphorine. Mutations at CYP2C9 residues 144 and 358 had little or no effect. Thus (a) wild type CYP2C19 and CYP2C9 are relatively low Km catalysts of cyclophosphamide and ifosfamide activation, and (b) all four human CYP2C enzymes activate these two anticancer prodrugs with varying efficiencies and with striking differences among naturally occurring allelic variants in the case of CYP2C9 and CYP2C18.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1997

Heterogeneity in androgen receptor levels and growth response to dihydrotestosterone in sublines derived from human hepatocellular carcinoma line (KYN-1).

Heterogeneity in the growth response to androgen and androgen receptor (AR) status in an AR-positive human hepatocellular carcinoma (HCC) line, KYN-1, was investigated in the present study. Seven sublines were obtained from KYN-1 by the limited dilution method. Four of them had no detectable amounts of AR and did not show any growth response to dihydrotestosterone (DHT) at the concentration up to 1000 nM, while the other three had varying amounts of both cytosolic and nucleosolic AR that were 5- to 7-fold higher than that of the parent cell. In the parent cell, the addition of DHT caused a modest but significant increase in the cell number (21%) in a 14-day culture. Such effect was increased by 2- to 7-fold in two AR-positive sublines. In addition, the sensitivity of the response in these two sublines was increased by up to 24-fold. The one remaining AR-positive subline did not show any growth response to DHT, although the behavior of its AR in sucrose gradient ultracentrifugation and Western blotting was the same as that of the other two sublines. These data clearly show that sublines derived from an established human HCC line have a broad heterogeneity in the growth response of HCC to androgen as well as in AR status and could be classified into three groups on the basis of the growth responsiveness to androgen and the AR expression.

Blotting, Western↗