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Biomedical subjects

L Yu

Publications and source records attributed to L Yu.

At least 73 records · Page 4Linked to original sources
PubMed2001

Identification of human, mouse and rat PPP1R14A, protein phosphatase-1 inhibitor subunit 14A, & mapping human PPP1R14A to chromosome 19q13.13-q13.2.

Three novel cDNAs encoding putative protein phosphatase-1 inhibitory protein (PPP1R14A) were isolated from human, mouse and rat. The human, mouse and rat PPP1R14A proteins were all of 147 amino acids sharing about 90% sequence identity to porcine CPI17, an inhibitor of protein phosphatase-1 holoenzyme. The protein sequence 33RHARVTVK40, which is important for the inhibitory potency of porcine CPI17, was conserved in mammalian PPP1R14A. Northern blot analysis showed that human PPP1R14A was highly expressed as a 600 bp transcript in heart, prostate, testis, ovary, colon, small intestine and pancreas, lowly in brain, placenta, skeletal muscle and spleen and lung. The distribution of mouse and rat PPP1R14A was more specific and different from that of human PPP1R14A, abundant in lung, moderate/abundant in testis, moderate in brain and low in heart. In addition, we mapped human PPP1R14A to chromosome 19q13.13-q13.2 by radiation hybrid mapping, and determined that the human PPP1R14A gene spanned a 5.1-kb region and consisted of four exons and three introns.

Amino Acid Sequence↗
PubMed2001

Augmentation of myocardial transfection using TerplexDNA: a novel gene delivery system.

Gene therapy is a potential new strategy for the treatment of cardiovascular disease. The most efficacious method of gene delivery remains a key hurdle to effective gene therapy. We present the application of a novel, nonviral gene delivery system (TerplexDNA) to augment myocardial transfection. The hearts of New Zealand white rabbits were injected with reporter genes, luciferase cDNA or beta-galactosidase cDNA, either as naked plasmid DNA or plasmid DNA complexed with stearyl-poly(L-lysine)-low density lipoprotein (TerplexDNA). Three day left heart myocardial cell lysates produced 44571 +/- 8730 RLU (RLU = total light units/mg protein) for the TerplexDNA luciferase rabbits versus 1638 +/- 567 RLU for the naked luciferase rabbits (P = 0.002). Thirty days after injection, myocardial lysates produced 677 +/- 52 RLU for the TerplexDNA luciferase hearts versus 18 +/- 3 RLU for the naked luciferase hearts (P = 0.002). Histologic analysis of the hearts transfected with beta-galactosidase showed that TerplexDNA increased the area and depth of transfection compared with the naked plasmid DNA alone. The hearts of Sprague-Dawley rats were injected in a similar fashion and analyzed at 1, 3, 5, 10, 15, 25 and 30 days after injection. The naked luciferase injected hearts showed transient elevation of luciferase activity to day 5 but fell back to baseline levels after that time-point. The TerplexDNA luciferase injected hearts had significantly elevated luciferase activity to 30 days. The Terplex gene delivery system significantly augments myocardial transfection compared with a naked plasmid DNA system alone. The advantage in transfection efficiency appears to be related to the unique properties of the TerplexDNA carrier molecule. The TerplexDNA delivery system represents a novel means to augment transfection of the myocardium.

Animals↗
PubMed2001

Detection of immunomagnetically captured Escherichia coli O157:H7 by antibody-conjugated alkaline phosphatase.

A rapid and sensitive detection process for Escherichia coli O157:H7 was developed using alkaline phosphatase (APase)-labeled anti-E. coli O157 antibodies to tag the targeted bacteria. Immunomagnetic beads or antibody-labeled streptavidin-coated magnetic beads were then used to capture the APase-tagged E. coli. Immunomagnetically captured bacteria were washed and distributed into microplates or optical cuvettes. The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed. Less than 1000 cfu/ml of E. coli O157:H7 could be detected. This approach was applied to detect the bacteria artificially spiked in beef hamburgers. Less than 1 cfu/g of E. coli O157:H7 produced a significant response after cultural enrichment for 4-6 h at 37 degrees C.

Alkaline Phosphatase↗
PubMed2001

Plasminogen activator inhibitor-1 gene polymorphism 4G/4G genotype and lupus nephritis in Chinese patients.

BACKGROUND: Abnormal regulation in the coagulation and fibrinolytic system may play an important role in mediating glomerular damage in lupus nephritis. Indeed, glomerular thrombosis occurs frequently in lupus nephritis and predicts the future development of glomerular sclerosis. In the murine model of active lupus nephritis, plasminogen activator inhibitor-1 (PAI-1) gene was overexpressed throughout the kidney, both within the glomeruli and also in tubules and vessels. The level of PAI-1 expression in the tissues appeared to correlate with the progression of lupus nephritis. Recently, a single base pair insertion/deletion 4G/5G polymorphism of the PAI-1 gene has been identified and shown to alter plasma PAI-1 activity. This study was therefore conducted to determine the association of the 4G/5G polymorphism of the PAI-1 gene with the development and severity of lupus nephritis. METHODS: The PAI-1 gene polymorphism of 118 systemic lupus erythematosus (SLE) patients and 103 healthy controls who were gender and age matched was determined using standard polymerase chain reaction. PAI-1 genotype results were studied in relationship to the development and severity of lupus nephritis. RESULTS: Allele frequencies of 4G/5G allele were 0.59/0.41 in lupus patients and 0.59/0.41 in controls (P = 1.000). No significant difference was noted in the genotype distribution between SLE patients with and without nephritis. However, lupus nephritis patients with the 4G4G genotype showed significantly heavier proteinuria (5.0 vs. 3.7 g/day; P = 0.023) when compared with patients with 4G5G and 5G5G genotypes. Also, 73.3% patients with 4G4G had an activity index > or =8 versus 37.3% patients with 4G5G and 5G5G (P = 0.003). Extensive necrotizing lesions were seen in 51.7% patients with 4G4G as compared with 23.5% patients with 4G5G and 5G5G (P = 0.014). The association of the 4G4G gene polymorphism with a higher nephritis activity and more severe necrotizing lesions persisted when only class III and class IV nephritis patients were studied. On the other hand, no significant association was noted between the PAI-1 gene polymorphism and the chronicity of the nephritis. CONCLUSION: These findings suggest that the 4G/5G polymorphism of the PAI-1 gene is associated with the activity but not the chronicity of lupus nephritis. The presence of the 4G4G genotype does not increase the risk of developing SLE or lupus nephritis, but predicts the development of higher nephritis activity and more extensive necrotizing lesions.

Adult↗
PubMed2001

Genetic variation in the hepatocyte nuclear factor (HNF)-3alpha gene does not contribute to maturity-onset diabetes of the young in Japanese.

Heterozygous mutations in the genes encoding transcription factors in the hepatocyte nuclear factor (HNF) cascade are associated with maturity-onset diabetes of the young (MODY), a monogenic form of diabetes mellitus. However, these genes are responsible for only approximately 20% of the cases of MODY in Japanese patients. Searching for a novel MODY gene in this population, we investigated a candidate for encoding the forkhead transcription factor HNF-3alpha, which also belongs to the HNF-transcription cascade. The human HNF-3alpha gene, which was assigned to the segment near microsatellites D14S75 and AFM200ZH4 on chromosome 14 by radiation hybrid mapping, spans approximately 5 kb and consists of two exons. Ninety-five Japanese subjects with MODY/early-onset non-ketotic diabetes were screened for mutations in this gene. Direct sequencing of the exons and flanking regions identified one missense mutation (Ala-83-Thr) in exon 2 and three nucleotide alterations in the non-coding regions. However, their frequencies were not significantly different between MODY and control subjects, indicating that mutations in the HNF-3alpha gene are not a major cause of MODY in Japanese patients.

DNA-Binding Proteins↗
PubMed2001

Laryngeal compartmentalization after radiation therapy in a canine model.

OBJECTIVE: We set out to study the effects of radiation therapy on laryngeal tissue barriers in a canine model as described by Welsh. STUDY DESIGN: India ink was injected into 1 vocal fold on irradiated and nonirradiated canine larynges. RESULTS: The india ink dye spread more prominently into the supraglottis of the irradiated specimens by submucosal lymphatics. Larger dye volumes simulating larger tumor bulk spread to the contralateral larynx in irradiated specimens. CONCLUSION: Conservation laryngeal surgery for laryngeal carcinoma is a popular method of treatment. Some surgeons advocate partial laryngectomy for radiation failures; in other instances, it is the primary modality of treatment. These findings suggest a cautious approach when planning conservative laryngeal surgery after radiation therapy failures of glottic carcinoma.

Animals↗
PubMed2001

Purification and characterization of homo- and hetero-dimeric acetate kinases from the sulfate-reducing bacterium Desulfovibrio vulgaris.

Two distinct forms of acetate kinase were purified to homogeneity from a sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F. The enzymes were separated from the soluble fraction of the cells on anion exchange columns. One acetate kinase (AK-I) was a homodimer (alpha(S)(2)) and the other (AK-II) was a heterodimer (alpha(S)alpha(L)). On SDS-PAGE, alpha(L) and alpha(S) subunits migrated as bands of 49.3 and 47.8 kDa, respectively, but they had an identical N-terminal amino acid sequence. A rapid HPLC method was developed to directly measure ADP and ATP in assay mixtures. Initial velocity data for AK-I and AK-II were collected by this method and analyzed based on a random sequential mechanism, assuming rapid equilibrium for the substrate binding steps. All kinetic parameters for both the forward acetyl phosphate formation and the reverse ATP formation catalyzed by AK-I and AK-II were successfully determined. The two enzymes showed similar kinetic properties in Mg(2+) requirement, pH-dependence and magnitude of kinetic parameters. These results suggest that two forms of acetate kinase are produced to finely regulate the enzyme function by post-translational modifications of a primary gene product in Desulfovibrio vulgaris.

Acetate Kinase↗
PubMed2001

Physicochemical consequences of the perdeuteriation of glutathione S-transferase from S. japonicum.

Glutathione S:-transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9 degrees C, whereas that for dGST was 51.0 degrees C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar K(m) to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.

Animals↗
PubMed2001

Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox.

Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X-linked chronic granulomatous disease by flow-cytometry. Therefore, we re-evaluated the location of the 7D5 epitope using gene-engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the approximately 58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycin-treated PLB-985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.

3T3 Cells↗
PubMed2001

Characterization of newly emerging Newcastle disease virus isolates from the People's Republic of China and Taiwan.

Seven Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken and pigeon flocks in China and Taiwan between 1996 and 2000 were genotypically and pathotypically characterized. By phylogenetic analysis of the fusion protein genes, isolates Ch-A7/96, Ch/98-3, Ch/99, Ch/2000, and TW/2000 were placed into two novel subgenotypes, VIIc and VIId. Isolate Ch/98-1 was grouped into subgenotype VIb, while Ch-W6/96 was proven to be a mixture of isolates Ch-A7/96 and Ch/98-1. These isolates were pathotyped as viscerotropic velogenic for Ch/98-3, Ch/99, Ch/2000, and TW/2000; neurotropic velogenic for Ch-A7/96; and mesogenic for Ch/98-1. Three separate, comparative, genetic analyses of the F genes, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, were performed with our isolates and selected NDV strains from GenBank. Results showed that the close genetic similarity provided evidence for the epidemiological linkage between the outbreaks in China and Taiwan and that the 1990s outbreaks in Asia, the Middle East, Africa, and Europe constituted the fourth panzootic of ND. In combination with epidemiological analysis, an evolutionary model of the NDV strains, representative of the direction of transmission within the NDV strains, was proposed, and epidemiology of NDV transmission was evaluated with emphasis on molecular aspects. Finally, a cross-protective experiment indicated that at least one strain (Ch-A7/96) among our NDV isolates was an antigenic variant, responsible for recent outbreaks of ND in vaccinated chicken flocks.

Animals↗
PubMed2001

Biostability and pharmacokinetics of LJP 920, an octameric Gal (alpha1-3) Gal conjugate for the inhibition of xenotransplantation rejection.

Antibodies to an alpha-galactosyl saccharide structure present in human serum are associated with hyperacute rejection and delayed xenograft rejection after pig-to-primate xenotransplantation. To overcome this major barrier to the xenotransplantation, LJP 920, a galactosyl alpha1-3 galactose (Gal (alpha1-3) Gal) coupled to a non-immunogenic platform at a valency of eight Gal (alpha1-3) Gal molecules/platform, was synthesized to clear circulating antibodies and to inhibit their production by B cells that produce these antibodies. Herein we report on the stability of UP 920 in biological media and its pharmacokinetic profile. Incubation of LJP 920 with mouse serum or liver microsomes at 37 degrees C for 2 days showed no indication of degradation of the conjugate as detected by a reversed-phase HPLC method, indicating that the conjugate is not subject to enzymatic metabolism. After intravenous administration of LJP 920 to mice at the doses of 20 and 100 mg kg(-1), UP 920 serum concentration decreased rapidly, showing a biphasic pattern, with a distribution half-life of 3 min and an elimination half-life of more than 30 min, respectively. The serum-to-erythrocyte concentration ratio of UP 920 was 33- and 36-fold excess at 0.5 and 5 min, respectively, after intravenous administration (100 mg kg(-1)). Both Cmax and AUC values increased in a dose-proportional manner. UP 920 displayed a great distribution to well-perfused tissues. It was eliminated mainly through renal excretion in the unchanged form, which accounted for 23% of the total amount within 8 h of dosing.

Animals↗
PubMed2001

Hepatosplenic gamma/delta T-cell lymphoma in immunocompromised patients. Report of two cases and review of literature.

We describe 2 male patients in whom hepatosplenic gamma/delta T-cell lymphoma (HSTL) developed 6 and 10 years after renal transplantation. The onset was abrupt with systemic symptoms, cytopenia, and hepatosplenomegaly. The histologic examination of the spleen (case 1), liver, and bone marrow revealed sinusoidal infiltrates of markedly abnormal lymphocytes. The neoplastic cells in these cases were CD2+, CD3+, CD4-, CD5-, CD7+, CD8+, CD16+, CD56+, beta F1-negative, and TIA-1-negative. Both cases displayed clonal rearrangement of the T-cell receptor (TCR) delta gene and the TCR beta gene. The spleen in case 1 was positive for Epstein-Barr virus genome and showed TCR-gamma gene rearrangement by polymerase chain reaction. Isochromosome 7 [i(7)(q10)] was found in each case. Both patients died within 4 months of diagnosis. HSTL has been reported in only 5 renal transplant recipients. HSTL may be relatively more frequent in immunocompromised patients compared with the general population.

Adult↗
PubMed2001

Iterative algorithm with a constraint condition for numerical reconstruction of a three-dimensional object from its hologram.

A novel method to obtain the three-dimensional mathematical model of a transparent object from its hologram is presented. The proposed method can numerically extract the object information from the fringe pattern of the hologram. Then an iterative algorithm is used to imitate an imaging system by focusing on different layers of the object; and by operating in both the spatial domain and the frequency domain, the algorithm produces a series of two-dimensional layer images. The object is finally reconstructed layer by layer. A constraint condition should be satisfied, and the noise distribution can be rearranged in different reconstruction cycles so as to get better reconstruction quality. Numerical simulations have proved the effectiveness of the proposed method.

Journal Article↗
PubMed2001

Peptide and major histocompatibility complex-specific breaking of humoral tolerance to native insulin with the B9-23 peptide in diabetes-prone and normal mice.

NOD mice spontaneously develop anti-insulin autoantibodies and diabetes. A dominant peptide recognized by T-cell clones from NOD mice is insulin B-chain peptide B9-23. When administered subcutaneously to NOD mice, this peptide decreases the development of diabetes. In this study, we evaluated the autoantibody response to native insulin after administration of the B9-23 peptide. In NOD mice, administration of the B9-23 peptide in incomplete Freund's adjuvant enhanced their insulin autoantibody response with a higher level and longer persistence. Induction of insulin autoantibodies with the B9-23 peptide was observed in non-diabetes-prone BALB/c mice and NOR mice within 2 weeks of administration, but this was not observed in C57BL/6 mice. A series of A-chain, other B-chain, and proinsulin peptides did not induce insulin autoantibodies. Induced anti-insulin autoantibodies could not be absorbed with the peptide alone but could be absorbed with native insulin. The B13-23 peptide (one of two identified epitopes within B9-23) when administered to BALB/c mice, induced autoantibodies, whereas peptide B9-16 did not. Induction of autoantibodies mapped to the major histocompatibility complex (MHC) rather than to the background genes. Both splenocytes with I-A(d)/I-E(d) or I-A(g7)/I-E(null) presented the B9-23 peptide to NOD islet-derived T-cell clones. Finally, administration of the B9-23 peptide to BALB/c mice, even without adjuvant, could induce insulin autoantibodies. Our results indicate that B-cell tolerance to intact insulin is readily broken with the presentation of the B9-23 insulin peptide, depending on the host's specific MHC.

Animals↗
PubMed2001

Expression of GAD65 and islet cell antibody (ICA512) autoantibodies among cytoplasmic ICA+ relatives is associated with eligibility for the Diabetes Prevention Trial-Type 1.

More than 71,000 relatives of type 1 diabetic patients have been screened for cytoplasmic islet cell antibodies (ICAs), GAD65 autoantibodies (GAAs), and ICA512 autoantibodies (ICA512AAs). Among those 71,148 relatives, 2,448 were cytoplasmic ICA+, and the remainder were ICA-. Of the ICA+ group, 1,229 (50.2%) were positive for GAAs and/or ICA512AAs. Among ICA- relatives, 1,897 (2.76%) were positive for GAAs and/or ICA512AAs. Given the large number of relatives positive for cytoplasmic ICA and negative for "biochemically" determined autoantibodies, and the converse, we analyzed the proportion of ICA+ relatives found eligible to participate in the intervention phase of Diabetes Prevention Trial-Type 1 (DPT-1). To be eligible for the parenteral insulin DPT-1 trial, a relative had to have first-phase insulin secretion below the 1st percentile of cut-points (for parents) or below the 10th percentile (for siblings and offspring). To be eligible for the oral insulin trial, a relative had to have first-phase insulin secretion above cut-points (>1st percentile for parents, >10th percentile for siblings/offspring) and be positive for anti-insulin autoantibodies. For both trials, DQB1*0602 was an exclusion criteria, cytoplasmic ICA positivity had to be confirmed, and an oral glucose tolerance test had to result in nondiabetic levels. Of 572 relatives found to be eligible for trial entry, 442 (77.3%) were positive for GAAs and/or ICA512AAs, although overall only 50.2% of ICA+ relatives were positive for GAAs and/or ICA512AAs. The positive predictive value for trial eligibility for ICA+ relatives with GAAs or ICA512AAs who completed staging was 51.0%. In contrast, only 11.9% of ICA+ but GAA- and ICA512AA- relatives were found to be eligible by DPT criteria for trial entry. Positivity for biochemically determined autoantibodies among cytoplasmic antibody-positive relatives is associated with eligibility for the DPT-1 study.

Adolescent↗
PubMed2001

[Cloning and characterization of pathogenesis-related protein 1 from wheat-H. villosa translocation lines].

A Triticum aestivum-Haynaldia villosa 6VS/6AL translocation line contained powdery mildew resistance gene Pm21 which is effective against all the current biotypes of Erygsiphe gramins. Cloning of the related genes for powdery mildew resistance is significance for understanding its resistance mechanism and disease resistance breeding. Using RT-PCR and RACE technology, a wheat pathogenesis related protein 1 cDNA clone (TaPr-1) corresponding to a mRNA differentially induced in resistant 6VS/6AL translocation line compared to susceptible wheat cultivar "Yangmai 5" by powdery mildew infection was isolated and characterized. This sequence contained 823 bp and had an open reading frame (ORF) containing 164 amino acids with 24 amino acids in the putative signal peptide and 140 amino acids comprising the mature peptide (15.1 kD). The deduced amino acid sequence showed close homology to PR-1 like proteins, which have been isolated from many plants. Northern blot analysis revealed the most abundantly accumulation of the corresponding mRNA 12 h after infection in translocation line (6VS/6AL). The obviously difference in the expression of the PRw-1 was also observed between resistant translocation line (6VS/6AL) and susceptible parent "Yangmai 5", it showed the TaPr-1 gene is related to powdery mildew resistance. Southern blot indicated that the wheat genome contains more than one copies of TaPr-1 genes, and there are polymorphism between translocation lines and "Yanmai 5", the result shows that 6VS may contain TaPr-1 genes.

Amino Acid Sequence↗
PubMed2001

Extracellular protons inhibit the activity of inward-rectifying potassium channels in the motor cells of Samanea saman pulvini.

The intermittent influx of K+ into motor cells in motor organs (pulvini) is essential to the rhythmic movement of leaves and leaflets in various plants, but in contrast to the K+ influx channels in guard cells, those in pulvinar motor cells have not yet been characterized. We analyzed these channels in the plasma membrane of pulvinar cell protoplasts of the nyctinastic legume Samanea saman using the patch-clamp technique. Inward, hyperpolarization-activated currents were separated into two types: time dependent and instantaneous. These were attributed, respectively, to K+ -selective and distinctly voltage-dependent K(H) channels and to cation-selective voltage-independent leak channels. The pulvinar K(H) channels were inhibited by external acidification (pH 7.8-5), in contrast to their acidification-promoted counterparts in guard cells. The inhibitory pH effect was resolved into a reversible decline of the maximum conductance and an irreversible shift of the voltage dependence of K(H) channel gating. The leak appeared acidification insensitive. External Cs (10 mM in 200 mM external K+) blocked both current types almost completely, but external tetraethylammonium (10 mM in 200 mM external K+) did not. Although these results do not link these two channel types unequivocally, both likely serve as K+ influx pathways into swelling pulvinar motor cells. Our results emphasize the importance of studying multiple model systems.

Biological Transport↗