Biomedical subjects

Z Tu

Publications and source records attributed to Z Tu.

At least 55 records · Page 3Linked to original sources

PubMed1998

Sphingosine-1-phosphate in cell growth and cell death.

Recent evidence suggests that branching pathways of sphingolipid metabolism may mediate either apoptotic or mitogenic responses depending on the cell type and the nature of the stimulus. While ceramide has been shown to be an important regulatory component of apoptosis induced by tumor necrosis factor alpha and Fas ligand, sphingosine-1-phosphate (SPP), a further metabolite of ceramide, has been implicated as a second messenger in cellular proliferation and survival induced by platelet-derived growth factor, nerve growth factor, and serum. SPP protects cells from apoptosis resulting from elevations of ceramide. Inflammatory cytokines stimulate sphingomyelinase, but not ceramidase, leading to accumulation of ceramide, whereas growth signals also leading to accumulation of ceramide, whereas growth signals also stimulate ceramidase and sphingosine kinase leading to increased SPP levels. We propose that the dynamic balance between levels of sphingolipid metabolites, ceramide, and SPP, and consequent regulation of different family members of mitogen-activated protein kinases (JNK versus ERK), is an important factor that determines whether a cell survives or dies.

Animals↗

PubMed1998

Expression and characterization of human glutamate-cysteine ligase.

Glutamate-cysteine ligase (GLCL) catalyzes the rate-limiting step in glutathione biosynthesis. GLCL comprises regulatory (GLCLR) and catalytic (GLCLC) subunits. To understand better the structure-function relationship of GLCL subunits and holoenzyme, human GLCLR and GLCLC genes were inserted into the baculovirus genome. Recombinant hGLCLR andhGLCLC were produced in cells infected with recombinant baculoviruses, and homogeneous hGLCL subunits and holoenzyme were purified from cell lysates with a Ni-NTA resin. Purified recombinant hGLCL holoenzyme was catalytically more active than hGLCLC with L-glutamate, L-alpha-aminobutyrate, and ATP as substrates. The selectivity of purified hGLCL holoenzyme for L-glutamate, L-alpha-aminobutyrate, or L-cysteine was significantly higher than for hGLCLC. Glutathione was a noncompetitive inhibitor for both hGLCL holoenzyme and hGLCLC. hGLCLC was more sensitive to inhibition by glutathione than hGLCL holoenzyme. Deletion of the first 25 amino acid residues at the amino terminus of GLCLC dramatically decreased GLCL activity, indicating that the amino terminus of GLCLC is required for full catalytic activity. Expressed and purified hGLCL provides a useful tool to investigate glutathione biosynthesis in vitro.

Animals↗

PubMed1998

Purification and characterization of rat kidney sphingosine kinase.

Sphingosine kinase catalyzes the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate, which plays important roles in numerous physiological processes, including growth, survival, and motility. We have purified rat kidney sphingosine kinase 6 x 10(5)-fold to apparent homogeneity. The purification procedure involved ammonium sulfate precipitation followed by chromatography on an anion exchange column. Partially purified sphingosine kinase was found to be stabilized by the presence of high salt, and thus, a scheme was developed to purify sphingosine kinase using sequential dye-ligand chromatography steps (since the enzyme bound to these matrices even in the presence of salt) followed by EAH-Sepharose chromatography. This 385-fold purified sphingosine kinase bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of 1 M NaCl. After concentration, the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and Superdex 75 gel filtration columns. Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa under denaturing conditions on SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is a monomer. Sphingosine kinase shows substrate specificity for D-erythro-sphingosine and does not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, DL-threo-dihydrosphingosine, or N,N-dimethylsphingosine. However, the latter two sphingolipids were potent competitive inhibitors. With sphingosine as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed Michaelis-Menten kinetics, with Km values of 5 and 93 microM for sphingosine and ATP, respectively. This study provides the basis for molecular characterization of a key enzyme in sphingolipid signaling.

Animals↗

PubMed1998

Up-regulation of glutamate-cysteine ligase gene expression by butylated hydroxytoluene is mediated by transcription factor AP-1.

Several regulatory elements, including AP-1 and NF-kappa B, are present in the 5'flanking region of the human glutamatex-cysteine ligase (EC 6.3.2.2, gamma-glutamyl-cysteine synthetase) catalytic subunit (GLCLC) gene. In this study, we investigated the role of redox-sensitive transcription factors in the regulation of GLCLC gene expression in LLC-PK1 cells that were exposed to the antioxidant butylated hydroxytoluene (BHT). Exposure of LLC-PK1 cells to 100 microM BHT induced expression of transcription factor AP-1, as demonstrated by an electrophoretic mobility shift assay. Peak AP-1 induction occurred after 3 h of incubation with BHT, BHT increased luciferase gene expression in cells that were transfected with a luciferase reporter vector containing an AP-1 element upstream of a SV40 promoter. Northern analysis showed that transcription of GLCLC gene in cells after incubation with BHT was increased 30% compared with control cells. Cellular glutathione concentrations were also significantly increased in cells exposed to BHT. In contrast, exposure of LLC-PK1 cells to 100 microM BHT did not alter expression of the transcription factor NF-kappa B. These results show that induction of transcription factor AP-1 by BHT is involved in transactivation of GLCLC gene expression.

Antioxidants↗

PubMed1998

Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response.

The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that influences growth and survival of yeast. Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G1 arrest and cell death. We now have identified a gene in Saccharomyces cerevisiae, LBP1, that regulates the levels of phosphorylated sphingoid bases and ceramide. LBP1 was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis. LBP1 and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity. In vitro characterization of Lbp1p shows that this phosphatase is Mg2+-independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine. The deletion of LBP1 results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of LBP1 and LBP2 results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.

Amino Acid Sequence↗

PubMed1998

Structural, genomic, and phylogenetic analysis of Lian, a novel family of non-LTR retrotransposons in the yellow fever mosquito, Aedes aegypti.

A retrotransposon named Lian-Aa1 was discovered in an intron of an AaHR3-1 gene of the yellow fever mosquito, Aedes aegypti. This retrotransposon contained a long open reading frame with 1,219 amino acids that included endonuclease, reverse transcriptase, and RNase H domains. It was shown that in the Rock strain of Ae. aegypti, there were up to 1,380 copies of Lian elements, equivalent to 0.8% of the entire genome. Five additional copies of Lian elements were isolated, mapped by restriction digestion, and partially sequenced. The 5' and 3' ends of the Lian family were determined by comparing the terminal sequences of the six copies and were subsequently confirmed by the identification of putative target duplications flanking Lian-Aa1 and Lian-Aa2. The Lian family is likely a novel family of non-long-terminal-repeat (non-LTR) retrotransposons that terminate in a repeat of (CTGA-TAC)2. On average, the six copies of Lian elements showed only 0.6% sequence divergence at the nucleotide level in both a 735-bp region at the 5' end and a 1,124-bp coding region. Genomic Southern blots also revealed a very high degree of similarity among hundreds of Lian elements, suggesting very recent activity of Lian. Furthermore, all six analyzed Lian elements were closely associated with one or more different families of repetitive elements. It is possible that these associations could reflect the complex relationship between Lian elements and the rest of the Ae. aegypti genome. Phylogenetic analyses based on the reverse transcriptase, domains of 36 non-LTR retrotransposons including Lian-Aa1 identified five major subgroups that were supported by bootstrap replications. In contrast to the majority of non-LTR retrotransposons, Lian-Aa1 has an RNase H domain that is similar to a few other non-LTR retrotransposons and some retroviruses, which is consistent with the previously proposed independent assortment of different domains during the evolution of retroelements.

Aedes↗

PubMed1998

The recognition of sentences in noise by normal-hearing listeners using simulations of cochlear-implant signal processors with 6-20 channels.

Sentences were processed through simulations of cochlear-implant signal processors with 6, 8, 12, 16, and 20 channels and were presented to normal-hearing listeners at +2 db S/N and at -2 db S/N. The signal-processing operations included bandpass filtering, rectification, and smoothing of the signal in each band, estimation of the rms energy of the signal in each band (computed every 4 ms), and generation of sinusoids with frequencies equal to the center frequencies of the bands and amplitudes equal to the rms levels in each band. The sinusoids were summed and presented to listeners for identification. At issue was the number of channels necessary to reach maximum performance on tests of sentence understanding. At +2 dB S/N, the performance maximum was reached with 12 channels of stimulation. At -2 dB S/N, the performance maximum was reached with 20 channels of stimulation. These results, in combination with the outcome that in quiet, asymptotic performance is reached with five channels of stimulation, demonstrate that more channels are needed in noise than in quiet to reach a high level of sentence understanding and that, as the S/N becomes poorer, more channels are needed to achieve a given level of performance.

Acoustic Stimulation↗

PubMed1998

Roles of sphingosine-1-phosphate in cell growth, differentiation, and death.

Recent evidence suggests that branching pathways of sphingolipid metabolism may mediate either apoptotic or mitogenic responses depending on the cell type and the nature of the stimulus. While ceramide has been shown to be an important regulatory component of apoptosis induced by tumor necrosis factor alpha and the Fas ligand, sphingosine-1-phosphate (SPP), a further metabolite of ceramide, has been implicated as a second messenger in cellular proliferation and survival induced by platelet-derived growth factor, neuronal growth factor, and serum. SPP protects cells from apoptosis resulting from elevations of ceramide. Inflammatory cytokines stimulate sphingomyelinase, but not ceramidase, leading to accumulation of ceramide, whereas growth signals also stimulate ceramidase and sphingosine kinase leading to increased SPP levels. We propose that the dynamic balance between levels of sphingolipid metabolites, ceramide, and SPP and consequent regulation of different members of the mitogen-activated protein kinases (JNK versus ERK) family is an important factor that determines whether a cell survives or dies.

Animals↗

PubMed1997

Enhancement of gene expression by transcription factor AP-1 is dependent on orientation of AP-1 element.

Transcription factor AP-1 induced by 12-O-tetradecanoylphorbol-13-acetate treatment of LLC-PK1 cells binds specifically to an AP-1 oligonucleotide. To study the effect of interaction of transcription factor AP-1 with its AP-1 element on gene expression, an AP-1 consensus sequence was cloned into a reporter vector. Expression of the reporter gene in transfected cells was greatly enhanced in the presence of the reverse-oriented AP-1 element upstream of a SV40 promoter. Mutation (G to C) of the central base in the AP-1 motif (TGAGTCA) abolished the enhancement for the reporter gene expression. These results indicate that the orientation of AP-1 element relative to the transcription machinery is important for its regulatory function. Moreover, sequences that flank the AP-1 motif are also required for its regulatory function.

Animals↗

PubMed1997

Three novel families of miniature inverted-repeat transposable elements are associated with genes of the yellow fever mosquito, Aedes aegypti.

Three novel families of transposable elements, Wukong, Wujin, and Wuneng, are described in the yellow fever mosquito, Aedes aegypti. Their copy numbers range from 2,100 to 3,000 per haploid genome. There are high degrees of sequence similarity within each family, and many structural but not sequence similarities between families. The common structural characteristics include small size, no coding potential, terminal inverted repeats, potential to form a stable secondary structure, A+T richness, and putative 2- to 4-bp A+T-biased specific target sites. Evidence of previous mobility is presented for the Wukong elements. Elements of these three families are associated with 7 of 16 fully or partially sequenced Ae. aegypti genes. Characteristics of these mosquito elements indicate strong similarities to the miniature inverted-repeat transposable elements (MITEs) recently found to be associated with plant genes. MITE-like elements have also been reported in two species of Xenopus and in Homo sapiens. This characterization of multiple families of highly repetitive MITE-like elements in an invertebrate extends the range of these elements in eukaryotic genomes. A hypothesis is presented relating genome size and organization to the presence of highly reiterated MITE families. The association of MITE-like elements with Ae. aegypti genes shows the same bias toward noncoding regions as in plants. This association has potentially important implications for the evolution of gene regulation.

Aedes↗

PubMed1997

Vitellin of the sweet potato whitefly, Bemisia tabaci: biochemical characterization and titer changes in the adult.

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4-20% native porelimiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem.

Animals↗

PubMed1997

Biochemical, molecular, and phylogenetic analysis of pyruvate carboxylase in the yellow fever mosquito, Aedes aegypti.

Pyruvate carboxylase (PC, pyruvate: carbon dioxide ligase [ADP-forming], EC 6.4.1.1) was purified from the yellow fever mosquito, Aedes aegypti. The purified PC showed two polypeptides of similar M(r) (133 and 128 k). The N-terminal sequences of both polypeptides were shown to be very similar, if not identical. A polyclonal antiserum against the 133 kDa polypeptide cross-reacted strongly with the 128 kDa polypeptide. PC was found in all tissues examined. Using a semi-quantitative Western blot assay, PC was shown to be concentrated in the indirect flight muscles and fat body preparations. The ratios of the 133 to 128 kDa polypeptides were shown to differ in various tissues and an Aedes albopictus cell line. The indirect flight muscle was the only tissue in which the 128 kDa polypeptide was more abundant, while both the midgut and the cell line showed almost exclusively the 133 kDa polypeptide. Both peptides were present in varying amounts in brain, malpighian tubule, ovary and fat body preparation. The two isoforms of PC could play different roles in the flight muscle and other tissues. Clones covering a complete cDNA of PC of A. aegypti were obtained using a directional approach. The 3952 bp nucleotide sequence, including a 3585 bp coding region, was determined from these cDNA clones. The deduced 1195 amino acid sequence has a calculated M(r) of 132,200. A putative mitochondrial targeting sequence was determined by comparing the deduced amino acid sequence to the N-terminal sequences of the mature protein. The presence of a mitochondrial targeting sequence indicates that the mosquito PC encoded by the cloned cDNA may be localized in the mitochondria. After the targeting sequence, three functional domains were identified in the following order; biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP). The mosquito PC showed very high similarity to PCs from other sources (55.1-75.2% identity). Genomic Southern analysis indicated that there could be two similar PC genes or a single PC gene with allelic polymorphism in the A. aegypti genome. The evolutionary relationship of PCs among different organisms was consistent with the accepted evolutionary relationship of their host organisms. The evolution of the domain structures of the biotin-dependent carboxylases including PC was also investigated. This analysis indicates that biotin-dependent carboxylases evolved from a common origin. The analysis also provides evidence for early gene duplication events that shaped the family of biotin-dependent carboxylases. Clear evidence for the coevolution of BC and BCCP domains is presented, although they are associated with very different CT domains and the relative position of the three functional domains varies between members of the biotin-dependent carboxylases.

Aedes↗

PubMed1997

[Construction of poliovirus cDNA chimera with (NKNDD)n and (NANP)n].

Oligonucleotides encoding one copy of NKNDD, containing a P.f. B epitope NKND, and two copies of CSP repeated B epitope NANP were synthesized. After self-ligation, NKNDD and (NANP)2 were cloned into the intermediate vector pSKMM. Series of clones with different copies of NKNDD and NANP were identified. (NKNDD)n/MM with three, four, five, eight copies of NKNDD and (NANP)n/MM with four, six copies of NANP were chosen. Then (NKNDD)n and (NANP)n of these recombinants were recovered respectively and finally inserted into the N-Ag I site of poliovirus type I Mahoney strain full-length cDNA, which is contained in the expression plasmid pSV202H+. As using semi-quantitative hybridization in identifying tranformants, the procedure in our experiment for cloning different copies of NKNDD and NANP was greatly simplified.

Animals↗

PubMed1997

[Surgical treatment of non-thoracotraumatic pneumomediastinum].

The experience of surgical treatment of non-thoracotraumatic pneumomediastinum in 48 cases was summarized. The etiology, classification, pathology, and surgical treatment of non-thoracotraumatic pneumomediastinum were discussed. We conclude that most non-thoracotraumatic pneumomediastinum was complicated by secondary diseases. Therefore, the effective treatments for primary diseases are important. A satisfactory effect must be based on different surgical techniques according to the different clinical symptoms and age of patients.

Adolescent↗

PubMed1996

[Surgical treatment of primary epicardial tumors].

Seven cases of primary epicardial tumors of 3200 cardiac operation cases were diagnosed definitely, including benign in 5, malignant in 2. Among them, readical resection was performed in 5 cases, partial resection in 1, and decompression of pericardium in 1. Data of this series were often regarded as tuberculous pericarditis or others incorrectly. UCC, CT and MRI were helpful in diagnosis. The authors emphasize early surgical treatment as soon as possible and using the different methods of operation whether to utilize cardiopulmonary bypass or not according to site, size and characteristics of tumors to resect tumors, if necessary, reconstruction the heart wall to maintain blood follow dynamics. The prognosis of the diseases was related to the pathology and the area of invasion of tumors.

Adolescent↗

PubMed1995

Analysis of a vitellogenin gene of the mosquito, Aedes aegypti and comparisons to vitellogenins from other organisms.

A genomic clone of the Aedes aegypti vitellogenin A1 gene was sequenced including 2015 bp of 5' untranscribed sequence, 6369 bp of open reading frame interrupted by two introns, and a short 3' untranslated region. Primer extension was used to identify the transcription initiation site. The amino termini of the large and small subunits were located by N-terminal sequencing of vitellin purified from eggs. The length of the signal sequence and the position of the cleavage site between the two subunits were also determined. Three sequential imperfect repeats were found near the beginning of the small subunit. The sequence of the coding region appears to be polymorphic. Comparison of the signal sequences of seven insect vitellogenin genes revealed several conserved leucines, and a conserved position of an intron. However, the signal sequences are not conserved between these genes and the yolk protein genes of Cyclorraphid Dipteran insects. The cleavage sites between the small and large subunits in the vitellogenins of the mosquito, A. aegypti, sawfly, Athalia rosae, boll weevil, Anthonomus grandis, and silkworm, Bombyx mori are flanked by sequences rich in serine. Pairwise dot matrix analysis at the protein level showed that the mosquito, boll weevil and silkworm vitellogenins are significantly related with approx. 50% similarity. One region of the three insect vitellogenin genes, near the N-terminal of the large subunit, showed the highest levels of similarity, from 57.5 to 64.4%. The position of cysteines in insect vitellogenins is conserved, particularly in the C-terminus of the large subunit. Dot matrix comparison of the mosquito vitellogenin with that of Xenopus laevis and Caenorhabditis elegans showed much lower, but still significant degrees of relationship. Pairwise comparisons of the mosquito vitellogenin and the Drosophila melanogaster yolk proteins did not show significant similarities. Potential regulatory regions in the mosquito VgA1 gene were identified by comparison to regulatory elements known from other organisms, especially D. melanogaster, which could provide useful information for further functional analysis.

Aedes↗

PubMed1995

Genetics of coxsackievirus B3 cardiovirulence.

The human enteroviruses, especially the coxsackie B viruses, have been established as aetiologic agents of human inflammatory heart disease, a condition which may lead to dilated cardiomyopathy and heart failure. It is clear from murine models of coxsackievirus B3-induced inflammatory heart disease that not all strains of the virus are cardiovirulent (able to cause disease). Here, we present preliminary data mapping the site in a coxsackievirus B3 genome which determines a cardiovirulent phenotype.

Animals↗

PubMed1995

The cardiovirulent phenotype of coxsackievirus B3 is determined at a single site in the genomic 5' nontranslated region.

We report the construction of chimeric coxsackievirus B3 (CVB3) strains in which sequences of an infectious cDNA copy of a noncardiovirulent CVB3 genome were replaced by the homologous sequences from a cardiovirulent CVB3 genome to identify which of 10 predicted genetic sites determine cardiovirulence. Cardiovirulent phenotype expression was consistently linked to nucleotide 234 (U in cardiovirulent CVB3 and C in avirulent CVB3) in the 5' nontranslated region. Reconstructions of the parental noncardiovirulent CVB3 genome from chimeras restored the noncardiovirulent phenotype when tested in mice. Inoculation of severe combined immunodeficient (scid) mice with the noncardiovirulent CVB3 strain resulted in massive cardiomyocyte necrosis in all animals. Sequence analysis of viral genomes isolated from twelve scid mouse hearts showed that only nucleotide position 234 was different (a C-->U transition) from that in the input parental noncardiovirulent CVB3 genome. Higher-order RNA structures predicted by two different algorithms did not demonstrate an obvious local effect caused by the C-->U change at nucleotide 234. Initial studies of parental and chimeric CVB3 replication in primary cultures of fetal murine heart fibroblasts and in adult murine cardiac myocytes demonstrated that viral RNA transcriptional efficiency is approximately 10-fold lower for noncardiovirulent CVB3 than for cardiovirulent CVB3. CVB3 did not shut off protein synthesis in murine cardiac fibroblasts, nor were levels of viral protein synthesis significantly different as a function of viral phenotype. Taken together, these data support a significant role for determination of the CVB3 cardiovirulence phenotype by nucleotide 234 in the 5' nontranslated region, possibly via a transcriptional mechanism.

Animals↗