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Biomedical subjects

M Yu

Publications and source records attributed to M Yu.

At least 145 records · Page 8Linked to original sources
PubMed1999

Identification of the 16S rRNA m5C967 methyltransferase from Escherichia coli.

The fmu gene product has been proposed to be an RNA methyltransferase [Koonin, E. V. (1994) Nucleic Acids Res. 22, 2476-2478]. Fmu has been cloned and expressed, and the encoded 47 kDa protein has been purified and characterized. The enzyme catalyzed specific methylation of C967 of unmodified 16S rRNA transcripts. A 16mer stem-loop structure containing C967 (nt 960-975) was also a good substrate for the enzyme in vitro. Methylation of C967 was confirmed by several methods including analysis of RNase T1 digests and nearest-neighbor analysis. Fmu did not catalyze methylation of transcripts of 23S rRNA. E. coli cells that contained kanr-disrupted fmu produced 16S rRNA that could be specifically methylated by Fmu in vitro at C967 but not C1407. Further, fmu disruption did not significantly alter the growth rate of E. coli in rich or minimal media. We propose renaming this ORF "rrmB" and the enzyme "RrmB" for rRNA methyltransferase.

5-Methylcytosine↗
PubMed1999

Effects of various amino acid 256 mutations on sarcoplasmic/endoplasmic reticulum Ca2+ ATPase function and their role in the cellular adaptive response to thapsigargin.

Upon direct selection of mammalian cells for resistance to thapsigargin (TG), a potent inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase (SERCA), the ATPase can acquire specific mutations at amino acid position 256 (aa256). In particular, Phe256 --> Leu and Phe256 --> Ser substitutions can occur upon TG selection, with each substitution resulting in a SERCA that is 4- to 5-fold resistant to TG inhibition (M. Yu et al., J. Biol. Chem. 273, 3542-3546, 1998). We have now identified a third substitution, i.e., Phe256 --> Val, that occurs when the Chinese hamster lung fibroblast cell line DC-3F is selected for TG resistance. Although the Phe256 --> Val substitution at codon 256 results in a SERCA whose enzymological properties in terms of Ca2+ transport and ATP hydrolysis are essentially similar to that of wild-type (wt) SERCA, the mutant enzyme is more than 40-fold resistant to TG inhibition. To analyze further the role of aa256 in TG-SERCA interactions, mutational analysis of this particular residue was also carried out. Of all the mutations introduced, only the Phe256 --> Glu substitution interferes with expression of the ATPase. The Phe256 --> Arg substitution does not interfere with SERCA expression, but the resulting enzyme is totally inactive. In terms of sensitivity of the various mutants to TG, maximal reduction in the ATPase's affinity for TG occurs with amino acid substitutions containing branched side chains, i.e. with the Phe256 --> Val, Phe256 --> Ile, and Phe256 --> Thr mutants. Since a corresponding Phe is conserved in the Na+, K+-ATPase which is not sensitive to TG, our findings suggest that this amino acid provides stabilization of the stalk segment with respect to the membrane interface, thereby optimizing specific interactions of TG with neighboring S3 residues (L. Zhong and G. Inesi, J. Biol. Chem. 273, 12994-12998, 1998). It is likely that a relatively high frequency of codon 256 mutations favor the aa256 mutants as a specific adaptive response to TG selection.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Th2 T cells expressing transgene PDGF-A serve as vectors for gene therapy in autoimmune demyelinating disease.

We hypothesized that T cells can be genetically modified to express growth factor transgene products capable of inducing oligodendrocyte progenitor proliferation. Autoreactive T cells isolated from SWXJ mice immunized with the p139-151 determinant of myelin proteolipid protein (PLP) were transfected with an antigen-inducible transgene for platelet-derived growth factor-A (PDGF), a growth factor important in regulating the development of oligodendrocytes. Isolated antigen-specific T cell clones expressed the PDGF transgene when stimulated with PLP 139-151 peptide and produced biologically active PDGF capable of inducing proliferation of oligodendrocyte progenitor cells. Furthermore, upon adoptive transfer, the PDGF transfected T cells migrated to the CNS and ameliorated ongoing disease. Our data indicate that autoreactive memory Th2 cells can be genetically modified so that upon engagement with self antigen they produce regenerative growth factors capable of mediating tissue repair during autoimmune disease.

Adoptive Transfer↗
PubMed1999

Regression and spreading of self-recognition during the development of autoimmune demyelinating disease.

The autoimmune T cell repertoire in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) is characterized by CD4(+)T cells of the Th1 phenotype that recognize peptide determinants of central nervous system (CNS) myelin proteins in an MHC class II-restricted manner. Our recent studies and those performed by others have shown that progression to chronicity in EAE and MS is accompanied by a broadening of the T cell repertoire with time. This acquired neo-autoreactivity is commonly referred to as epitope spreading and is presumably the result of endogenous priming to new self-determinants during the CNS inflammation that accompanies disease onset and relapse. In the present study we extend our earlier observations by showing that disease progression in both EAE and MS is accompanied by two concurrent events, viz. (1) the spontaneous regression of the primary established autoimmune repertoire associated with disease onset, and (2) the emergence of the epitope spreading cascade associated with disease progression. Our data show that disease initiation and disease progression in both EAE and MS are typically associated with distinctly different autoimmune T cell repertoires. Our data support the view that the natural development of self-recognition during autoimmune disease may best be understood when considered in the temporal context of an 'epitope du jour' and 'moving target' perspective.

Amino Acid Sequence↗
PubMed1999

Purification, characterization, and immunolocalization of hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase from opium poppy.

A development-specific and elicitor-inducible acyltransferase [hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110)] that catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to hydroxyphenethylamines was purified 988-fold to apparent homogeneity from opium poppy (Papaver somniferum L.) cell-suspension cultures. The purification procedure, which resulted in a 6.8% yield, involved hydrophobic interaction and anion-exchange chromatography, followed by affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent. Purified THT had an isoelectric point of 5.2, a native molecular mass of approximately 50 kDa, and consisted of two apparently identical 25-kDa subunits as determined by two-dimensional polyacrylamide gel electrophoresis. The purified enzyme was able to synthesize a variety of amides due to a relatively low specificity for cinnamoyl-CoA derivatives and hydroxyphenethylamines. The best substrates were feruloyl-CoA (VK(m)(-1)13.4 mkat g(-1) M(-1)) and tyramine (VK(m)(-1)6.57 mkat g(-1) M(-1)). The THT activity increased during development of opium poppy seedlings, occurred at high levels in roots and stems of mature plants, and was induced in cell-suspension cultures after treatment with a pathogen-derived elicitor. Immunoblot analysis using THT mouse polyclonal antibodies did not always show a correlation between THT polypeptide and enzyme activity levels. For example, despite low THT activity in leaves, an abundant 25-kDa immunoreactive polypeptide was detected. Immunohistochemical localization showed that THT polypeptides occur in cortical and xylem parenchyma, immature xylem vessel elements, root periderm, anthers, ovules, and the inner layer of the seed coat, but are most abundant in phloem sieve-tube members in roots, stems, leaves, and anther filaments.

Acyltransferases↗
PubMed1999

Total peripheral conductance mediates antihypertensive effect of nonpeptide mixed endothelin receptor antagonist in deoxycorticosterone acetate-salt hypertensive rats.

The relative contribution of cardiac output (CO) and total peripheral conductance (TPC) to the changes in blood pressure (BP) evoked by an intravenous injection of bosentan, a nonpeptide mixed endothelin (ET) antagonist, were investigated in conscious unrestrained deoxycorticosterone acetate (DOCA)-salt hypertensive rats and sham-control rats. Blood pressure was recorded by radiotelemetry devices and CO by ultrasonic transit-time flow probes. Bosentan significantly reduced BP (from 141 +/- 3 to 111 +/- 3 mm Hg) and increased TPC (from 1.19 +/-0.1 to 1.72 +/- 0.2 mL/min/kg/mm Hg) in DOCA-salt hypertensive rats, but not in sham rats. An increase in CO opposed the BP-lowering effect of the antagonist. The results demonstrate that the role of ET receptors in the maintenance of the hypertensive state in the DOCA-salt model of hypertension is exerted at the level of the resistance vessels and not on factors that regulate cardiac output.

Animals↗
PubMed1999

Effect of radiochemical purity of 99Tcm-Q12 on myocardial SPET image quality.

In myocardial perfusion SPET studies with 99Tcm-Q12, we observed that some patients had high liver uptake that interfered significantly in the assessment of the inferior wall. The aim of this study was to assess the effects of the radiochemical purity of 99Tcm-Q12 on liver uptake. Thirty-one patients undergoing routine myocardial infarction perfusion studies were evaluated. The radiochemical purity of 99Tcm-Q12 was determined using HPLC. Venous blood samples taken 50 min after injection of 99Tcm-Q12 during peak exercise were also analysed. Liver uptake was expressed as the liver-to-heart ratio. In addition, the SPET images were classified by two experienced nuclear medicine specialists into three groups representing high-quality images (n = 7), images with high general background activity (n = 13) and images with high liver and/or intestinal uptake (n = 11). The liver-to-heart ratio correlated inversely with the radiochemical purity of 99Tcm-Q12 (r = -0.65, P < 0.001) and unchanged 99Tcm-Q12 in plasma (r = -0.44, P < 0.02). The radiochemical purity of 99Tcm-Q12 was significantly lower in the group with high liver uptake (60.1 +/- 4.2%) than in the group with good-quality images (81.8 +/- 5.6%, P < 0.01) or with high background activity (82.3 +/- 2.5%, P < 0.01). In conclusion, the radiochemical purity of 99Tcm-Q12 has a significant inverse correlation with the liver-to-heart ratio; thus, the high radiochemical purity of 99Tcm-Q12 should be confirmed to prevent interference by liver uptake.

Chromatography, High Pressure Liquid↗
PubMed1999

Decreased cell wall digestibility in canola transformed with chimeric tyrosine decarboxylase genes from opium poppy

Tyrosine decarboxylase (TYDC) is a common plant enzyme involved in the biosynthesis of numerous secondary metabolites, including hydroxycinnamic acid amides. Although a definite function has not yet been determined, amides have been proposed to form a physical barrier against pathogens because they are usually found as integral cell wall components. Canola (Brassica napus) was independently transformed with chimeric genes (35S::TYDC1 and 35S::TYDC2) under the transcriptional control of the cauliflower mosaic virus 35S promoter, and encoding two TYDC isoforms from opium poppy (Papaver somniferum). All T0 plants displayed a suppressed level of wild-type TYDC activity, and transgene mRNAs were not detected. Silencing of 35S::TYDC1 was overcome in the T1 progeny of self-pollinated T0 plants, since high levels of TYDC1 mRNAs were detected, and TYDC activity increased up to 4-fold compared with wild-type levels. However, TYDC1 mRNA levels decreased in T2 plants and were not detected in the T3 progeny. TYDC activity also gradually declined in T2 and T3 plants to nearly wild-type levels. In contrast, silencing of 35S::TYDC2 was maintained through four consecutive generations. T1 plants with a 3- to 4-fold increase in wild-type TYDC activity showed a 30% decrease in cellular tyrosine pools and a 2-fold increase in cell wall-bound tyramine compared with wild-type plants. An increase in cell wall-bound aromatic compounds was also detected in these T1 plants by ultraviolet autofluorescence microscopy. The relative digestibility of cell walls measured by protoplast release efficiency was inversely related to the level of TYDC activity.

Journal Article↗
PubMed1999

Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast.

Cell cycle G2 arrest, nuclear localization, and cell death induced by human immunodeficiency virus type 1 Vpr were examined in fission yeast by using a panel of Vpr mutations that have been studied previously in human cells. The effects of the mutations on Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, induction of cell cycle G2 arrest by Vpr was found to be independent of nuclear localization. In addition, G2 arrest was also shown to be independent of cell killing, which only occurred when the mutant Vpr localized to the nucleus. The C-terminal end of Vpr is crucial for G2 arrest, the N-terminal alpha-helix is important for nuclear localization, and a large part of the Vpr protein is responsible for cell killing. It is evident that the overall structure of Vpr is essential for these cellular effects, as N- and C-terminal deletions affected all three cellular functions. Furthermore, two single point mutations (H33R and H71R), both of which reside at the end of each alpha-helix, disrupted all three Vpr functions, indicating that these two mutations may have strong effects on the overall Vpr structure. The similarity of the mutant effects on Vpr function in fission yeast and human cells suggests that fission yeast can be used as a model system to evaluate these Vpr functions in naturally occurring viral isolates.

Cell Death↗
PubMed1999

Functional, biochemical, and molecular investigations of renal kallikrein-kinin system in diabetic rats.

A reduction of renal kallikrein has been found in non-insulin-treated diabetic individuals, suggesting that an impaired renal kallikrein-kinin system (KKS) contributes to the development of diabetic nephropathy. We analyzed relevant components of the renal KKS in non-insulin-treated streptozotocin (STZ)-induced diabetic rats. Twelve weeks after a single injection of STZ, rats were normotensive and displayed hyperglycemia, polyuria, proteinuria, and reduced glomerular filtration rate. Blood bradykinin (BK) levels and prekallikrein activity were significantly increased compared with controls. Renal kallikrein activity was reduced by 70%, whereas urinary BK levels were increased up to threefold. Renal kininases were decreased as indicated by a 3-fold reduction in renal angiotensin-converting enzyme activity and a 1.8-fold reduction in renal expression of neutral endopeptidase 24.11. Renal cortical expression of kininogen and B2 receptors was enhanced to 1.4 and 1. 8-fold, respectively. Our data suggest that increased urinary BK levels found in severely hyperglycemic STZ-diabetic rats are related to increased filtration of components of the plasma KKS and/or renal kininogen synthesis in combination with decreased renal kinin-degrading activity. Thus, despite reduced renal kallikrein synthesis, renal KKS is activated in the advanced stage of diabetic nephropathy.

Animals↗
PubMed1999

Development of an In Vitro Screening Assay for Compounds that Increase Bone Formation.

Although parathyroid hormone (PTH) has the ability to stimulate bone growth in both rats and humans, its mechanism of action is not fully understood at the molecular level. An in vitro marker that reflects the in vivo anabolic actions of PTH would facilitate the discovery of small-molecule compounds that stimulate bone growth. We therefore compared the patterns of gene expression in three cell lines treated with PTH. The levels of c-fos, collagenase, interleukin-6 (IL-6), and collagen mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) in three osteoblast-like cell lines. The most responsive marker was c-fos, which was induced 5-10-fold after 1 h of PTH treatment in the UMR106-01 cell line. Because it is a critical early response gene in bone growth, we investigated the possibility of using c-fos stimulation as a method to screen for compounds that can stimulate bone formation. A highly sensitive, medium-throughput RT-PCR assay for c-fos mRNA expression was established using the Taqmantrade mark Detection System (Perkin Elmer, Mississauga, Ontario). Cells were treated with a series of compounds to determine the specificity of c-fos stimulation. Of the compounds tested, only PTH, prostaglandin E(2), 8-bromo-cAMP, and forskolin induced c-fos mRNA levels, indicating that this assay was specific for compounds that are known to induce cAMP and stimulate bone growth. These results indicate that a simple in vitro assay for c-fos may be a reliable method for the screening of compounds that stimulate bone growth in vivo.

Journal Article↗
PubMed1999

[Haplotype identification of single sperm in pigs].

Haplotype of porcine single sperm was analyzed by PCR method through using primer extension preamplification and heminesting primer design strategy. The PCR products were run on 8% polyacrylamide gel, and visualized by silver staining method. The results showed that the haplotypes of individual sperm can be unequivocally demonstrated by these methods. The application of single sperm typing provides a unique tool for the construction of high resolution genetic map and the study of genetic phenomena requiring large sample size.

Animals↗
PubMed1999

Therapeutic efficacy of transcatheter arterial embolization of primary hepatocellular carcinoma: discrepancy in different histopathologic subtypes.

OBJECTIVE: To evaluate preliminarily the therapeutic efficacy of transcatheter arterial embolization (TAE) for different histopathologic subtypes of primary hepatocellular carcinoma (HCC). METHODS: A retrospective study of 226 patients with histopathologically diagnosed primary HCC was performed. The patients were treated with either single TAE, surgical resection of tumor alone, or TAE combined with surgical resection. Follow-up information was achieved in 157 of 226 patients. Comparative analyses of survival data and image findings were performed with correlation to histopathologic classification and different therapeutic methods, respectively. RESULTS: Eight histopathologic subtypes of primary HCC were found in this group, including HCC of trabecular pattern, pseudoglandular pattern, fibrolamellar HCC and sclerosing HCC, as well as HCC of clear cell, of small cell, poorly differentiated or undifferentiated HCC, and hormonally active HCC. The accumulated survival rate for these 157 patients was 74.52% of 1 year, 53.50% of 2 years, 31.85% and 14.01% of 3 and 5 years, respectively. Fibrolamellar HCC and clear cell HCC had relatively higher survival rate (25.00% and 33.22% of 5 years, respectively) than that of other subtypes, and the median survival time of the latter was 71 months. The mean survival time was 25.06 months (SE = 1.87) in single TAE group, 30.38 (SE = 2.05) months in surgical resection, and 72 months (SE = 6.90) in TAE combined with resection. CONCLUSIONS: Discrepancies do exist in therapeutic effect of different subtypes of HCC. In this study, clear cell HCC was more sensitive to TAE than other subtypes, and, in contrast, small cell HCC and poorly differentiated or undifferentiated HCC were of lower sensitivity to TAE.

Carcinoma, Hepatocellular↗
PubMed1999

[The effects of levothyroxine replacement therapy on bone and mineral metabolism in patients with hypothyroidism].

OBJECTIVE: To investigate the effects of short-term thyroid hormone replacement therapy on bone and mineral metabolism in patients with hypothyroidism. METHODS: Enzyme linked immunosorbent assay (ELISA) was used to measure urinary deoxypyridinoline (DPD) and radioimmunoassay (RIA) to measure serum calcitonin (CT), parathyroid hormone (PTH-M), bone GLA protein (BGP), free T(3) (FT(3)), free T(4) (FT(4)) and thyroid-stimulating hormone (TSH) in 29 patients with hypothyroidism before and after treatment with levothyroxine for (11.5 +/- 2.5) months. Dual energy X-ray absorptiometry was used to measure bone mineral density (BMD) of their spine (L(2 - 4)) and femur neck, trochanter and Ward's triangle. The results were compared with those in 37 healthy controls. RESULTS: In hypothyroidism patients before treatment, serum BGP level was significantly lower (P < 0.05) than but urinary DPD was similar with those of control. BMD was significantly lower in postmenopausal patients (P < 0.01 - 0.001) than that in control group, but there was no change in premenopausal patients. After replacement therapy serum BGP and urinary DPD elevated significantly (P < 0.05), whereas BMD decreased slightly both in the premenopausal and postmenopausal patients when compared with that before treatment, with no statistical significance (P > 0.05). BMD decreased by 0.7% and 1.7% - 5.1% in premenopausal and postmenopausal patients respectively, but there was no significant difference between these two groups (P > 0.05). FT(3) was positively correlated with BGP. FT(4) and FT(3) were positively correlated with DPD. BMD of femur neck and trochanter sites was negatively correlated with FT(4). CONCLUSION: Short-term physiological dose levothyroxine replacement therapy causes increase of bone turnover and leads to bone mass loss to various extent on hypothyroidism patients.

Adult↗
PubMed1999

[Intestinal bacterial translocation in patients with biliary tract diseases].

To determine whether intestinal bacterial translocation occurs in man, the authors cultured the tissues of mesenteric lymph nodes(MLNs), bile, portal and peripheral venous blood obtained prior to and during operation in 35 patients with biliary diseases. No positive peritoneal swab, portal blood and peripheral blood culture were shown. Thirteen of 20 patients with biliary obstruction (65%) had bacteria in their MLNs, while no positive MLN culture was found in 12 patients without biliary obstruction. The most common bacteria recovered from the MLNs were Gram-negative enteric bacilli. Thus, it is considered that extrahepatic biliary obstruction may induce intestinal bacterial translocation in man.

Adult↗
PubMed1999

[The properties of new affinity adsorbent for adsorbing the cycle immune complex in blood].

This study was designed to select an affinity adsorbent from 5 kinds of adsorbents available. The adsorbent AA3 was found being specific to the adsorption of pathologic cycle immune complex (CIC). The physical properties such as specific surface area, proe volume and average pore diameter of adsorbents were determined. A discussion on the factors that affect the adsorption of CIC is also presented in this paper.

Adsorption↗
PubMed1999

[Optimization of fermentation conditions for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Alcaligenes eutrophus].

A close relationship, between the initial addition time and concentration of propionate and HV fraction, was observed in shaking culture of Alcaligenes eutrophus for the production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate). The optimal initial addition time of propionate was determined at the onset of PHBV formation period. Although relatively high HV unit could be obtained under high propionate concentration, the growth and product synthetic activities were inhibited obviously. Different ratios of glucose to propionate were fed to stimulate the formation of HV unit and the results were compared. The optimizing feeding strategy of propionate was proposed based on the consideration of several fermentation index. The final cell dry weight, PHB concentration, PHB content and HV fraction in PHBV reached 52.1 g/L, 40.8 g/L, 78.3% and 16.2 mol%, respectively, Yield coefficient of HV unit to propionate and PHBV productivity were obtained to be 0.5 g/g and 0.74 g/(L/h), respectively.

Cupriavidus necator↗