Biomedical subjects

T Li

Publications and source records attributed to T Li.

At least 37 records · Page 2Linked to original sources

PubMed1999

The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity.

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.

Acylation↗

PubMed1999

A neuronal-specific mammalian homolog of the Drosophila retinal degeneration B gene with expression restricted to the retina and dentate gyrus.

Mutations in the Drosophila retinal degeneration B (rdgB) gene cause a rapid loss of the electrophysiological light response and subsequent light-enhanced photoreceptor degeneration. The rdgB gene encodes a protein with an N-terminal phosphatidylinositol transfer protein domain, a large C-terminal segment, and several hydrophobic regions thought to multiply span the subrhabdomeric cisternal membrane. A mammalian rdgB homolog (m-rdgB1) was previously identified and shown to exhibit widespread tissue distribution and functionally rescue the Drosophila rdgB mutant phenotypes. We describe a second mammalian rdgB homolog (m-rdgB2) that possesses 46% amino acid identity to Drosophila RdgB and 56% identity to M-RdgB1. M-RdgB2 possesses a neuronal-specific expression pattern, with high levels in the retina and the dentate gyrus mossy fibers and dendritic field. Using M-RdgB2-specific antibodies and subcellular fractionation, we demonstrate that M-RdgB2 is not an integral membrane protein but is stably associated with a particulate fraction through protein-protein interactions. Although transgenic expression of M-RdgB2 in rdgB2 null mutant flies suppressed the retinal degeneration, it failed to fully restore the electrophysiological light response. Because transgenic expression of M-RdgB2 does not restore the wild-type phenotype to rdgB2 mutant flies to the same extent as M-RdgB1, functional differences likely exist between the two M-RdgB homologs.

Amino Acid Sequence↗

PubMed1999

-141 C del/ins polymorphism of the dopamine receptor 2 gene is associated with schizophrenia in a British population.

Dopamine has long been hypothesised to be involved in the pathogenesis of schizophrenia. The dopamine D2 receptor is a major site of action of neuroleptic agents used in the treatment of schizophrenia. Arinami et al. [1997; Human Mol Genet 6:577-582] have recently sequenced the dopamine receptor 2 (DRD2) gene in Japanese individuals and identified a novel polymorphism: a single cytosine deletion at position -141 disrupting a BstN1 restriction site with a frequency of 0.22 in their control group. They then found a strong association with this polymorphism and schizophrenia (p < 0.001) with an odds ratio of 0.60 in a Japanese population. We have attempted to verify their results by repeating the RFLP analysis on a sample of Scottish schizophrenics and controls. We then combined our data with those from another British sample recruited using similar procedures. The total combined sample size was 439 schizophrenics and 437 controls. We obtained a significant association--p = 0.02 with an odds ratio of 1.41. Schizophrenia is associated with the C insertion in the Japanese, but that association is reversed in Caucasians. Linkage disequilibrium with a causative polymorphism nearby is the most likely explanation for this reverse association.

Alleles↗

PubMed1999

Discrimination of tRNALeu isoacceptors by the insertion mutant of Escherichia coli leucyl-tRNA synthetase.

A variant (LeuRS-A) of Escherichia coli leucyl-tRNA synthetase (LeuRS) carrying a 40-residue duplication in its connective peptide 1 (CP1) has a 3-fold lower specificity for than for, whereas wild-type LeuRS has the same specificity for these two isoacceptors. The replacement of the acceptor stem of with yields a chimeric tRNA(Leu) for which wild-type LeuRS has the same specificity as it does for the two normal isoacceptors mentioned, but for which LeuRS-A has a reduced specificity similar to that for, indicating a difference between these two acceptor stems. LeuRS-A is slightly less stable than the native enzyme. Wild-type LeuRS and LeuRS-A have almost same K(d) value for their interaction with as determined by fluorescence quenching. No difference was detected between these two proteins by CD and fluorescence spectroscopy. These results show that LeuRS-A can discriminate between the two isoacceptors of tRNA(Leu).

Acylation↗

PubMed1999

Association analysis between dopamine receptor genes and bipolar affective disorder.

We have performed a case-control analysis of dopamine D2-like receptor (DRD2, DRD3 and DRD4) gene polymorphisms in 118 Han Chinese cases with bipolar affective disorder and 196 control subjects, and replication analysis in 157 English cases and 143 control subjects. We found association between a functional DRD2 promoter variant (P = 0.03 by allele) and the DRD2 taq1A polymorphism (P = 0.001 by allele) in Chinese bipolar disorder patients. However, this finding was not replicated in the Caucasian subjects, indicating that the significant association we observed in the Chinese population is a false positive finding. An alternative explanation is that these polymorphisms are risk factors in Chinese but not Caucasian populations, a hypothesis which seems unlikely in view of the similarity of the clinical characteristics of bipolar disorder in the two populations. We also report a novel, rare one-repeat variant of the DRD4 exon 3 VNTR repeat in Chinese populations, which appears to be absent in Caucasians and is not associated with disease.

Adult↗

PubMed1999

Apoptosis in isolated adult cardiomyocytes exposed to adriamycin.

Adriamycin (doxorubicin) is a highly potent antineoplastic agent, but its use is limited by the risk of developing cardiomyopathy and congestive heart failure. Available evidence suggests that adriamycin-induced congestive heart failure is mediated by oxidative stress. We examined the possibility of adriamycin-induced apoptosis in isolated adult rat cardiomyocytes and its inhibition by trolox, a water-soluble antioxidant. Cardiomyocytes isolated from rat hearts were exposed to 20 microM adriamycin for 1 h and examined at different post-treatment durations (0-23 h). Adriamycin caused a significant decrease in rod-shaped cells and an increase in round cells. Both Hoechst 33258 staining and TUNEL assay revealed a significantly increased number of apoptotic myocytes and nucleosomal fragmentation upon exposure to adriamycin. In agarose gel electrophoresis, DNA laddering was found to be more intense in adriamycin-exposed myocytes. A bright smear at the leading edge of the gels suggested indiscriminate fragmentation of DNA and myocyte necrosis by adriamycin. Both types of DNA degradations due to adriamycin were significantly reduced by trolox. We suggest that adriamycin-induced cell death involves both apoptosis and necrosis and these may be mediated by oxidative stress.

Animals↗

PubMed1999

Ethnic variation in the thymidylate synthase enhancer region polymorphism among Caucasian and Asian populations.

Thymidylate synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A tandem repeat sequence in a TS promoter enhancer region (TSER) was recently identified. Polymorphic variation affected in vitro expression levels of the gene. We evaluated the influence of ethnicity on TSER genotype. Allele frequency was similar in Caucasian and Southwest Asian subjects. However, homozygous triple repeat subjects were twice as common in Chinese subjects (67%) than in Caucasian subjects (38%). This demonstrates significant ethnic variation in a TS gene regulatory element which may have significant impact on pyrimidine homeostasis and drug therapy.

Alleles↗

PubMed1999

Rescue of cyclin D1 deficiency by knockin cyclin E.

D-type cyclins and cyclin E represent two very distinct classes of mammalian G1 cyclins. We have generated a mouse strain in which the coding sequences of the cyclin D1 gene (Ccnd1) have been deleted and replaced by those of human cyclin E (CCNE). In the tissues and cells of these mice, the expression pattern of human cyclin E faithfully reproduces that normally associated with mouse cyclin D1. The replacement of cyclin D1 with cyclin E rescues all phenotypic manifestations of cyclin D1 deficiency and restores normal development in cyclin D1-dependent tissues. Thus, cyclin E can functionally replace cyclin D1. Our analyses suggest that cyclin E is the major downstream target of cyclin D1.

Animals↗

PubMed1999

Lack of reciprocal genomic imprinting of sense and antisense RNA of mouse insulin-like growth factor II receptor in the central nervous system.

Two models have been proposed to account for the molecular mechanism underlying genomic imprinting of the insulin-like growth factor II receptor gene (Igf2r): expression-competition and promoter DNA methylation. To examine which model best explains the regulation of Igf2r imprinting, we examined the allelic expression of endogenous Igf2r sense and antisense RNAs in mice. In peripheral tissues, Igf2r sense and antisense RNAs show a reciprocal pattern of imprinting and DNA methylation between the two parental alleles: the sense RNA is monoallelically expressed only from the maternal promoter which is unmethylated in region 1, and the antisense RNA is derived solely from the paternal promoter which is unmethylated in region 2. The paternal promoter of sense Igf2r and the maternal promoter of antisense Igf2r are hypermethylated and are transcriptionally suppressed. In CNS, the genomic imprinting of Igf2r sense and antisense RNAs is uncoupled: both parental promoters of Igf2r gene coding for sense RNA are unmethylated and are biallelically used for transcription. In contrast, antisense RNA of Igf2r is derived only from the paternal allele that is unmethylated in region 2, while the methylated maternal allele is silent. Uncoupling of genomic imprinting of Igf2r sense and antisense RNAs in CNS correlates with DNA methylation of the appropriate promoter region, thus favoring the model of DNA methylation over that of antisense as the chief regulator of Igf2r genomic imprinting.

Alleles↗

PubMed1999

Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development.

The vertebrate lens is a tissue composed of terminally differentiated fiber cells and anterior lens epithelial cells. The abundant, preferential expression of the soluble proteins called crystallins creates a transparent, refractive index gradient in the lens. Several transcription factors such as Pax6, Sox1, and L-Maf have been shown to regulate lens development. Here we show that mice lacking the transcription factor c-Maf are microphthalmic secondary to defective lens formation, specifically from the failure of posterior lens fiber elongation. The marked impairment of crystallin gene expression observed is likely explained by the ability of c-Maf to transactivate the crystallin gene promoter. Thus, c-Maf is required for the differentiation of the vertebrate lens.

Aging↗

PubMed1999

The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein beta subunit.

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein alpha subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Here we report that functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein beta subunit (Gbeta5L). RGS9 and Gbeta5L also form a complex when coexpressed in cell culture. Our data are consistent with the recent observation that several RGS proteins, including RGS9, contain G protein gamma-subunit like domain that can mediate their association with Gbeta5 (Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. & Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. USA 95, 13307-13312). We report an example of such a complex whose cellular localization and function are clearly defined.

Amino Acid Sequence↗

PubMed1999

[Long-term follow-up of combined therapy with large-dose zinc sulfate and low-dose penicillamine in children with hepatolenticular degeneration].

OBJECTIVE: To summarize the long-term effect of combined treatment with large-dose zinc sulfate and low-dose penicillamine in children with hepatolenticular degeneration (HLD). METHODS: The patients who had symptoms were treated with large-dose zinc sulfate (100-150mg, <6yr; 150-200mg, 6-8yr; 200-300mg, 9-10yr; 300mg,>10yr; 3 times a day) in addition to low-dose penicillamine(8-10mg/kg/d) at the beginning of treatment. Zinc sulfate alone was given to the presymptomatic patients and it was used as maintenance therapy when clinical improvement was obtained. 31 children were followed up for 4-11 years. RESULTS: In 3 presymptomatic patients, no clinical abnormalities were found. Among 28 patients with symptoms, 23 patients (82%) had their symptoms and signs subsided or much improved, 2 patients(7%) remained unchanged, and 3(11%) died. Blood concentrations of copper were persistently lower than normal. Urine copper excretion of 24 hours was significantly lower than that before the combined therapy in all patients, and it became normal in 5 cases(16%) after 6 months of treatment, and in 26 cases(84%) after 1-2 years of treatment. Higher blood concentrations of zinc were found in 20 cases(65%), and higher urine zinc excretion was noted in 25 cases(81%) once or more times during the therapy. CONCLUSION: Combined therapy of large-dose zinc sulfate and low-dose penicillamine is an effective, safe and cheap treatment for children with HLD.

Adolescent↗

PubMed1999

102T/C polymorphism of serotonin receptor type 2A gene is not associated with schizophrenia in either Chinese or British populations.

Several pieces of evidence implicate serotonin receptors in the aetiology of schizophrenia, and recently a number of studies have reported a genetic association between the 102T/C polymorphism of serotonin receptor type 2A gene and schizophrenia. Unfortunately a number of failures to replicate these positive associations in both Caucasian and Chinese populations have also been reported. We have examined the 102T/C polymorphism by PCR amplification and restriction analysis of DNA from: 202 schizophrenics and 202 controls from Shanghai; 112 schizophrenics and 224 parents from Chengdu, Cina; and 253 schizophrenics and 244 controls from the the UK. We find no evidence of association or transmission disequilibrium between the 102T/C polymorphism and schizophrenia in any of the groups we have examined. We conclude that either the original positive reports occurred by chance or any effect must be minimal, and urge caution in interpreting small positive results derived using data from different centres.

Carrier Proteins↗

PubMed1999

Recombinant rat fibroblast growth factor-16: structure and biological activity.

Fibroblast growth factor-16 (FGF-16) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-16 (rFGF-16) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA into Escherichia coli for high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, during E. coli fermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-16, expressed the gene in E. coli, and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-16 extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of beta-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-16 induces oligodendrocyte proliferation in vitro, and induces hepatocellular proliferation and increased liver weight in vivo. In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties.

3T3 Cells↗

PubMed1999

Direct injection of plasma to determine pseudoephedrine by high performance liquid chromatography with column switching.

An HPLC method has been developed for the determination of pseudoephedrine in plasma using column switching. Preparation of the sample was simple in that only 1000 microL of water was added to 200 microL of plasma before injection. A 900 microL aliquot was injected onto the precolumn. Double distilled water was used to elute and remove proteins and polar components in the sample. The components retained on the precolumn were flushed forward onto the analytical column by the mobile phase (acetonitrile-0.2 mol/L ammonium sulphate, 10:90 v/v) with automated column switching. The limit of determination of pseudoephedrine in plasma was 12 ng/mL. The relative standard deviations of intra- and inter-assay for the determination of pseudoephedrine in plasma were 1.2-9.8% over the concentration range 1020-21.8 ng/mL. The mean recovery by on-line solid phase extraction was 94.76% (RSD = 1.1%).

Calibration↗

PubMed1999

Accuracy and sensitivity of DNA pooling with microsatellite repeats using capillary electrophoresis.

DNA pooling is a genetic screening method that combines DNA from many individuals in a single polymerase chain reaction (PCR) reaction to generate a representation of allele frequencies. The substantial saving in effort with DNA pooling over individual genotyping facilitates linkage disequilibrium scanning of the human genome using many thousands of genetic markers, and is applicable to mapping of complex diseases such as schizophrenia. However, the literature to date has not addressed several crucial technical aspects of DNA pooling. These include: DNA quantification; the choice of electrophoresis methods; sensitivity (the minimum reliably detectable difference between pools); and methods of dealing with 'plus-A' stutter. We have examined these points and make recommendations as to the best procedures to adopt as well as quantifying reproducibility and sensitivity. We conclude that, although allele frequencies derived from microsatellite pooling are distorted, differences of 5% or greater between pools can be reliably detected.

Alleles↗

PubMed1999

The correlation between R2' and bone mineral measurements in human vertebrae: an in vitro study.

The aim of this study was to investigate whether MR imaging of trabecular bone structure using magnetic inhomogeneity measurements is related to the amount of bone mineral in human vertebrae. Weight, bone mineral content (BMCDXA), bone mineral per area (BMADXA) and bone mineral density (BMDCT) were determined in 12 defatted human lumbar vertebrae (L2-L4) by weighing, dual X-ray absorptiometry (DXA) and CT. Inhomogeneity caused by susceptibility differences between trabecular bone and surrounding water was studied with MR imaging at 1.5 T using the GESFIDE sequence. The pulse sequence determines the transverse relaxation rate R2(*) and its two components, the non-reversible transverse relaxation rate (R2) and the reversible transverse relaxation rate (R2'; i. e. relaxation rate due to magnetic susceptibility) in a single scan. Voxel size was 0.9 x 1.9 x 5.0 mm. Positive significant correlations between R2' and weight, BMCDXA, BMADXA and BMDCT were observed (r > 0.61 and p < 0.05 for all). Unexpectedly, R2 was also positively correlated with weight, BMCDXA and BMDCT (r > 0.66 and p < 0.05 for all), but not with BMADXA. Thus, R2' measurements are related to the amount of bone mineral, but they also provide information which is not obtainable from bone mineral measurements.

Absorptiometry, Photon↗

PubMed1999

Bilateral orbital emphysema from compressed air injury.

PURPOSE: To describe a patient who developed bilateral subconjunctival and orbital emphysema after an automobile tire explosion. METHOD: Case report. RESULTS: A 60-year-old man sustained bilateral ocular injury after a tire explosion. Ophthalmic examination disclosed bilateral subconjunctival air, with no visible conjunctival laceration. Computed tomography showed orbital emphysema, with no evidence of orbital fracture. Follow-up examination 2 weeks after the injury disclosed resolution of the subconjunctival air. Best-corrected visual acuity in the right eye was decreased after the explosion but improved to the baseline level of 20/40 2 weeks after the injury. CONCLUSION: Subconjunctival and orbital emphysema can occur from high-pressure air injury in the absence of an obvious entry site.

Air↗