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Biomedical subjects

J Xu

Publications and source records attributed to J Xu.

At least 991 records · Page 55Linked to original sources
PubMed1995

Baculovirus-mediated production of the human growth hormone in larvae of the silkworm, Bombyx mori.

To express the cDNA encoding human growth hormone (hGH) in larvae of Bombyx mori, B. mori nuclear polyhedrosis virus (BmNPV) was employed as an expression vector. For the construction of the recombinant virus, the hGH cDNA was inserted into the downstream of the strong polyhedrin promoter to achieve a high level expression. Immunoblot analysis revealed that the virus-mediated hGH was synthesized in the larvae and secreted into the hemolymph. The yield of the recombinant hGH synthesized in the larvae reached to a level of 160 micrograms/ml of hemolymph after purification. The purified recombinant hGH was confirmed to have both the same molecular weight and amino acid sequence at its N-terminal region as those of the natural counterpart. In addition, the biological activity of the recombinant hGH was comparable to that of the natural hGH in the growth-stimulating effect on rat Nb 2 Node lymphoma cells.

Animals↗
PubMed1995

Fractal dimension of exon and intron sequences.

In this paper, the concept of fractal is applied to describe the features of nucleotide sequences. We introduce the mapping from nucleotide sequences to two-dimensional metric space. Then we use this mapping to study quantitatively the self-similarity of exon and intron sequences in different scales. We find that self-similarity exists in the geometrical range and main range of a nucleotide sequence and define the fractal dimension in these ranges. The results show that the fractal properties of exon sequences are quite different from those of introns, reflecting their difference in structure and function. The fractal dimension of the geometrical range may be used to predict the exon regions of a raw nucleotide sequence.

Animals↗
PubMed1995

Quantitative determination of serum iron in human blood by high-performance capillary electrophoresis.

A capillary electrophoretic (HPCE) method that can be used to quantitatively determine trace amounts of iron has been developed and applied to determine the iron level in human serum. After precipitation of serum proteins, Fe(III) in the serum is reduced to Fe(II) with hydroxylamine hydrochloride, and a stable Fe(II)-1,10-phenanthroline complex is formed by adding 1,10-phenanthroline to the supernatant containing 2.5 mM ammonium acetate-acetic acid at pH 5.0. The Fe(II)-1,10-phenanthroline complex, [Fe(C12H8N2)3]2+, has a very strong absorbance at 270 nm (with a molar absorptivity of approximately 9.2.10(4)). By measuring the absorbance of [Fe(C12H8N2)3]2+ at 270 nm, the iron level in human serum can be precisely quantified. The interference from copper, a major interference in serum, can be totally eliminated due to the complete separation of [Fe(C12H8N2)3]2+ and the Cu(II)-1,10-phenanthroline complex. In addition, other problems that usually occurred with conventional spectrophotometric methods, such as co-precipitation and occlusion of iron during sample pretreatment, are significantly minimized due to the ability to wash the precipitate and the higher detection sensitivity. With this method, a single drop (10 microliters) of serum would be sufficient to determine the serum iron concentration. The method is reliable, sensitive, rapid and reproducible. Thus it is highly suitable for use in the clinical laboratory.

Electrophoresis, Capillary↗
PubMed1995

Specific sequestering agents for the actinides. 28. Synthesis and initial evaluation of multidentate 4-carbamoyl-3-hydroxyl-1-methyl-2(1H)-pyridinone ligands for in vivo plutonium(IV) chelation.

A new family of chelating agents based on 4-(substituted-carbamoyl)-3-hydroxy-2-pyridinones is reported. These have optional terminal substituents on the nitrogens, and the hydroxypyridonate (HOPO) rings are attached to molecular backbones through amide linkages. A very important feature of the methyl-substituted ligand derivatives (Me-3,2-HOPOs) is that, similarly to the catechoylamide complexes of the siderophore enterobactin and its analogs, these HOPO derivatives form strong hydrogen bonds between the amide proton and the adjacent oxygen of the phenolate in the metal complex; this enhances the stability of the complex. This rigidity helps to explain the great affinity of the Me-3,2-HOPO ligands for plutonium(IV), as observed here under physiological conditions. All 13 compounds studied significantly enhanced Pu excretion from mice compared with Pu-injected controls. Eight of the ligands studied promoted significantly more Pu excretion than an equal molar amount of CaNa3-DTPA (the compound in present clinical use). Five injected and two orally administered Me-3,2-HOPO ligands promoted as much or slightly more Pu excretion than an equal molar amount of the octadentate 3,4,3-LI(1,2-HOPO), the previously most effective in vivo ligand. Surprisingly, although plutonium has an eight-coordination requirement, tetra- and hexadentate Me-3,2-HOPO ligands were essentially as effective as the one octadentate ligand studied. These observations suggest that even the tetradentate Me-3,2-HOPO ligands compete with mammalian transferrin for Pu(IV). For the three most promising compounds, there is no acute toxicity seen up to the highest dose administered, which was 1000 mumol/kg. One compound, the hexadentate TREN-(Me-3,2-HOPO), is particularly effective, either injected or orally, and an exceptionally good in vivo chelator of several actinides in addition to Pu(IV). Three of these compounds studied have low toxicity and are relatively simple and inexpensive to prepare. They are promising therapeutic agents.

Administration, Oral↗
PubMed1995

Stereochemistry-dependent bending in oligonucleotide duplexes induced by site-specific covalent benzo[a]pyrene diol epoxide-guanine lesions.

The apparent persistence length of enzymatically linearized pIBI30 plasmid DNA molecules approximately 2300 bp long, as measured by a hydrodynamic linear flow dichroism method, is markedly decreased after covalent binding of the highly tumorigenic benzo[a]pyrene metabolite 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. In striking contrast, the binding of the non-tumorigenic, mirror-image 7S,8R,9R,10S enantiomer [(-)-anti-BPDE] to DNA has no measurable effect on its alignment in hydrodynamic flow gradients (< or = 2.2% of the DNA bases modified). In order to relate this effect to BPDE-nucleotide lesions of defined stereochemistry, the bending induced by site-specifically placed and stereochemically defined (+)- and (-)-anti-BPDE-N2-dG lesions in an 11mer deoxyoligonucleotide duplex was studied by ligation and gel electrophoresis methods. Out of the four stereochemically isomeric anti-BPDE-N2-deoxyguanosyl (dG) adducts with either (+)-trans, (-)-trans, (+)-cis, and (-)-cis adduct stereochemistry, only the (+)-trans adduct gives rise to prominent bends or flexible hinge joints in the modified oligonucleotide duplexes. Since both anti-BPDE enantiomers are known to bind preferentially to dG (> or = 85%), these observations can account for the differences in persistence lengths of DNA modified with either (+)-anti-BPDE or the chiral (-)-anti-BPDE isomer.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Ligand-specific structural domains in the fibroblast growth factor receptor.

Two tandem immunoglobulin-like disulfide loops (Loops II and III) linked by a short connecting sequence in the ectodomain of the fibroblast growth factor receptor kinase compose the binding sites for glycosaminoglycan and fibroblast growth factor (FGF) ligands. Alternate splicing of exons IIIb and IIIc coding for the COOH-terminal half of Loop III confers high affinity for FGF-7 or FGF-2, respectively, on the fibroblast growth factor receptor ectodomain without effect on the binding of FGF-1. Here we show that a 139-amino acid fragment composed of Loop II, the inter-Loop II/III sequence, and a short segment of the NH2 terminus of Loop III is sufficient and near the minimal requirement for binding of FGF-1, FGF-2, and FGF-7. Extension of the fragment by five additional highly conserved residues (SD(P/A)QP) within a distinct constitutive structural domain (fl1) in Loop III restricts the binding of FGF-7 without effect on FGF-1 and FGF-2. Since the presence of exon IIIc in the full-length ectodomain does not change this ligand binding profile, we suggest that alternately spliced exon IIIc plays no active role in binding of the three ligands. In contrast, exon IIIb actively abrogates the restriction on the binding of FGF-7 and concurrently lowers the affinity for FGF-2.

Alternative Splicing↗
PubMed1995

Alternately spliced NH2-terminal immunoglobulin-like Loop I in the ectodomain of the fibroblast growth factor (FGF) receptor 1 lowers affinity for both heparin and FGF-1.

Alternate splicing of a single exon encoding an NH2-terminal immunoglobulin (Ig) disulfide loop in the ectodomain of the fibroblast growth factor receptor (FGFR) types 1 and 2 results in alpha and beta isoforms that exhibit 3- and 2-Ig loops, respectively. Previously we demonstrated that alternately spliced Loop I has no independent ligand binding activity but is sufficiently interactive with the ligand- and heparin-binding site formed by Loops II and III to lower affinity for the same fibroblast growth factor (FGF) ligand. Here we show that a lower affinity of FGFR1 alpha for heparin parallels the lower affinity for FGF-1. A mutant of FGFR1 alpha in which the sequence between Loops I and II was deleted exhibits high affinity for both FGF-1 and heparin and other properties of the FGFR1 beta isoform, which include resistance to degradation by trypsin and display of specific antibody epitopes. This suggests that the interloop sequence facilitates the interaction of Loop I with Loops II and III. Lack of expression of both exons coding for Loop I and the sequence between Loops I and II in the FGFR2 gene characterizes rat prostate tumor cells, which exhibit a loss of the low affinity class of FGF receptors. Although the exon coding for the sequence between Loops I and II is alternately spliced in the FGFR2 beta isoform, coordinate expression with the exon coding for Loop I results in the functional differences between the FGFR alpha and FGFR beta variants.

Alternative Splicing↗
PubMed1995

Induction of major histocompatibility complex class II antigen on amoeboid microglial cells in early postnatal rats following intraperitoneal injections of lipopolysaccharide or interferon-gamma.

The induction of major histocompatibility complex (MHC) class II antigen on amoeboid microglia cells (AMC) by lipopolysaccharide (LPS) and interferon-gamma (INF-gamma) in early postnatal rat brain was studied by immunohistochemistry. In newborn rats given successive intraperitoneal injections of LPS or INF-gamma and killed at the age of 7 days, MHC class II antigen expressing AMC were consistently present in specific areas throughout the entire brain, notably in the subcortical white matter and circumventricular region. It is concluded from this study that the induction of MHC class II antigen on AMC by LPS or INF-gamma is a widespread phenomenon in the developing brain. Since MHC class II antigen is essential for the initiation of immune response, it is suggested that besides their phagocytic nature, AMC may also be involved in immunological processes in the developing brain.

Animals↗
PubMed1995

Cloning, sequencing and expression of the pyrophosphate-dependent phosphofructo-1-kinase from Naegleria fowleri.

The cDNA for the PPi-dependent phosphofructo-1-kinase has been cloned and sequenced from a cDNA library prepared from the free-living amoeba Naegleria fowleri. The coding sequence of the cDNA consists of 1311 bases which translates into 437 amino acids with a molecular mass of 48095 Da. Comparison of the sequence with those of the previously described sequences of PPi-dependent phosphofructokinases from Propionibacterium freudenreichii and potato tuber revealed amino acid identities of 23 and 28% respectively and high conservation in those regions assumed to be part of the active site. The reading frame was cloned into an expression vector, which was transformed into Escherichia coli. Extracts of the transformed cells contained PPi-dependent phosphofructokinase activity that could be purified to homogeneity. The activity was lost on incubation with the chaotropic agent, KSCN, and recovered by subsequent incubation with AMP. These properties are consistent with those described by Mertens, De Jonckheere and Van Schaftingen [Biochem. J. (1993) 292, 797-803] for the enzyme prepared from Naegleria and support the idea that the cloned cDNA coded for the complete native enzyme. No nucleotide-binding motif or evidence for a nucleotide-binding site characteristic of the ATP-dependent phosphofructokinases could be found within the primary structure.

Adenosine Monophosphate↗
PubMed1995

Inhibin antagonizes inhibition of liver cell growth by activin by a dominant-negative mechanism.

The beta:beta activin homodimer and alpha:beta inhibin heterodimer are mutual antagonists which share a common beta subunit. Recently, it has been shown that, similar to transforming growth factor-beta 1, activin is an inhibitor of hepatocyte DNA synthesis. The activin receptor appears to be an obligatory complex of genetically distinct type I and II transmembrane serine/threonine kinases. Activin type I receptors, SKR1 and SKR2, were first cloned from well differentiated human hepatoma cells (HepG2). This prompted us to investigate the binding of activin and inhibin to receptors from HepG2 cells and the effect of the two ligands on DNA synthesis. Here we show that beta:beta activin binds to the activin type II receptor kinase (ActRII) which induces activin binding to the type I receptor kinase SKR2 to form ActRII.beta:beta.SKR2 complexes in which an activin beta chain occupies each receptor subunit. Inhibin also binds to ActRII through its beta subunit, competes with the binding of activin to ActRII, but fails to form the ActRII.SKR2 complex. No specific binding site for inhibin could be demonstrated in HepG2 cells. Inhibin, which had no activity of its own, antagonized the inhibitory effect of activin on DNA synthesis. The results suggest that inhibin may be a natural antagonist of assembly of the heterodimeric activin receptor complex through a dominant-negative mechanism.

Activin Receptors↗
PubMed1995

The N terminus of phosducin is involved in binding of beta gamma subunits of G protein.

Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the beta gamma subunits of guanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated phospholipase C beta 2 isoform activation. Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function. Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to G beta gamma. In contrast, the C-terminal half failed to bind to G beta gamma. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue.

Amino Acid Sequence↗
PubMed1995

Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury.

Levels of calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir) in the lumbar dorsal spinal cord of rats with either sciatic nerve transection or chronic constriction injury (CCI) were measured using radioimmunoassay. Significant decreases in CGRP-ir and SP-ir occurred in the ipsilateral spinal cord at 10 and 31 days after nerve transection. An ipsilateral decrease in SP-ir occurred 60 days after CCI. In addition, contralateral decreases in CGRP-ir and SP-ir occurred 31 days after transection and 60 days after CCI. Transection of the sciatic nerve produced greater decreases in peptide levels than did the CCI. Changes in spinal levels of these peptides may be involved in the appearance of neuropathic signs associated with nerve injury.

Animals↗
PubMed1995

Cloning and expression of glucocorticoid-induced genes in fetal rat lung fibroblasts. Transforming growth factor-beta 3.

Glucocorticoids have been shown to accelerate fetal lung type II cell maturation, and this effect appears, in part, to be mediated via fibroblasts. To identify glucocorticoid induced genes in fetal lung fibroblasts, we screened a cDNA library from cortisol-treated fetal lung fibroblasts with a subtracted cDNA probe which was enriched for sequences specific for cortisol-treated fetal lung fibroblasts. Fifty-seven clones were isolated from the cDNA library. One cDNA represented approximately 30% of the 57 clones. Analysis of DNA sequence homology suggested that this cDNA encodes the rat transforming growth factor-beta 3 (TGF beta 3). We found that TGF beta 3 mRNA was expressed in fetal lung fibroblasts but not epithelial cells. Expression of message in fetal lung fibroblasts was developmentally regulated. TGF beta 3 mRNA levels were low during the pseudoglandular stage (day 18), peaked during the early canalicular stage of lung development (day 19), then fell again at days 20 and 21 (term = 22 days). Exposure of fetal lung fibroblasts to cortisol increased TGF beta 3 mRNA expression in a time- and dose-dependent manner. Maternal administration of dexamethasone also enhanced mRNA expression of TGF beta 3 in fetal lung fibroblasts. These data suggest that glucocorticoids may mediate their stimulatory effect on lung maturation by inducing TGF beta 3 expression in fetal lung fibroblasts.

Amino Acid Sequence↗
PubMed1995

Regulation of mRNA levels for pulmonary surfactant-associated proteins in developing rabbit lung.

Gene transcriptional activities and steady-state mRNA levels have been examined for the surfactant-associated proteins SP-A, SP-B and SP-C in developing rabbit lung. It was observed SP-C mRNA levels increase early in gestation, while SP-A and SP-B mRNA levels increase rapidly between 26 and 30 days gestation. Transcriptional activities for all three surfactant apoproteins increase between 26 and 30 days. Studies conducted with fetal lung explants of 26 days gestation demonstrated exposure to low doses of dexamethasone increases SP-A and SP-C mRNA levels, while high doses stimulate transcription, although this only significant for SP-C. Time course studies revealed different temporal patterns and glucocorticoid responses for SP-A and SP-C mRNAs. SP-A and SP-C mRNA production and steady-state levels were reduced after treatment with cycloheximide. In contrast, SP-B gene transcription was selectively stimulated, suggesting involvement of a labile negative regulatory factory. It is concluded that expression of the three surfactant apoproteins is independently regulated. Early in gestation, SP-C mRNA levels may be regulated in vivo through message stabilization. Glucocorticoids can affect SP-A and SP-C mRNA levels in culture at both transcriptional and post-transcriptional levels. The ability of glucocorticoids to influence these processes declines during fetal development.

Animals↗
PubMed1995

Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori.

Nitric oxide (NO) synthase activity was detected in fat body and the Malpighian tubles of the silkworm, Bombyx mori. Main NO synthase activity in the fat body was Ca(2+)/calmodulin-dependent, inducible by bacterial lipopolysaccharide (LPS) and required NADPH, FAD, FMN, dithiothreitol (DTT) and tetrahydrobiopterin (BH4) as cofactors for the full expression of the activity. The Malpighian tubles contained two types of NO synthase. One was Ca(2+)-independent, calmodulin-dependent and constitutive and the other was Ca(2+)-dependent and constitutive. The former NO synthase required the same cofactors as fat body NO synthase. The activity of Malpighian tuble NO synthases increased dramatically at the end of the last instar period, just prior to spinning. These results indicate that B. mori contains new types of NO synthase, suggesting the wide distribution and different characteristics of this enzyme among vertebrates and invertebrates.

Amino Acid Oxidoreductases↗
PubMed1995

Construction of a transcription map surrounding the BRCA1 locus of human chromosome 17.

We have used a combination of methods (exon amplification, direct selection, direct screening, evolutionary conservation, island rescue-PCR, and direct sequence analysis) to survey approximately 600 kb of genomic DNA surrounding the BRCA1 gene for transcribed sequences. We have cloned a set of fragments representing at least 26 genes. The DNA sequence of these clones reveals that 5 are previously cloned genes; the precise chromosomal location of 2 was previously unknown, and 3 have been cloned and mapped by others to this interval. Three other genes, including BRCA1 itself, have recently been mapped independently to this region. Sequences from 11 genes are similar but not identical matches to known genes; 5 of these appear to be the human homologues of genes cloned from other species. Another 7 genes have no similarity with known genes. In addition, 39 putative exons and 14 expressed sequence tags have been identified and mapped to individual cosmids. This transcript map provides a detailed description of gene organization for this region of the genome.

Animals↗