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Biomedical subjects

B Lu

Publications and source records attributed to B Lu.

At least 199 records · Page 11Linked to original sources
PubMed1996

Expression of synapsin I correlates with maturation of the neuromuscular synapse.

Synapsins are a family of neuron-specific phosphoproteins that are localized within the presynaptic terminals in adult brain. Previous work has demonstrated that introduction of exogenous synapsins I(a + b) or IIa into Xenopus spinal neurons promoted maturation of the neuromuscular synapse in a nerve-muscle co-culture system. We have now studied the expression of endogenous Xenopus synapsin I during synaptic maturation in vivo and in culture, using a polyclonal antibody raised against Xenopus synapsin I. Immunoprecipitation experiments indicated that synapsin I was not detectable during the early phase of synaptogenesis in vivo, and exhibited a marked increase during the period of synaptic maturation. In contrast, the expression of synaptophysin, another synaptic vesicle protein, was detected at the start of nervous system formation, and remained at a high level thereafter. Similar expression profiles for the two proteins were also observed in immunocytochemical studies of Xenopus spinal neurons in culture: intense staining of synaptophysin was found on the first day, while synapsin I was not detected until after three days in culture. The expression of synapsin I correlated very well with the appearance of a bell-shaped amplitude distribution of spontaneous synaptic currents, a physiological parameter which reflects functional maturation of the neuromuscular synapse. In one-day-old cultures grown in the absence of laminin, an extracellular matrix protein known to be present at the neuromuscular junction, the amplitude distribution of virtually all synapses was skewed towards smaller values. In contrast, when laminin was used as a culture substrate, many synapses exhibited a bell-shaped amplitude distribution. Laminin treatment also induced synapsin I expression in one-day-old cultures. These results suggest that the expression of endogenous synapsin I may regulate maturation at neuromuscular synapses.

Animals↗
PubMed1996

Oriented immobilization of antibodies and its applications in immunoassays and immunosensors.

The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed. Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined.

Antibodies↗
PubMed1996

In vivo wear of three types of metal on metal hip prostheses during two decades of use.

Wear was analyzed on 21 metal on metal hip replacements, including McKee-Farrar, Müller, and Ring, that were retrieved from patients after as many as 25 years. Light and scanning electron microscopy indicated that early wear included substantial third body abrasion, possibly from particles generated while scratches from the original polishing were being eradicated and from dislodged surface carbides. However, the main contact zones were eventually worn smoother than the original surfaces. Wear was quantified by digitizing the shapes of the components on a coordinate measuring machine and identifying those areas that deviated from the original spheric surface. On the femoral heads, wear was typically concentrated in the superomedial region, that is, on the load axis. Three cases also had substantial wear inferiorly, but there were no cases with circumferential (equatorial) wear. The long term wear rates averaged approximately 6 micrometers per year or less and produced an average of approximately 6 mm3 of metallic wear debris per year or less. Wear rate tended to increase as clearance increased over the range of 127 to 386 micrometers, and a McKee-Farrar prosthesis with the extreme clearance of 1.7 mm wore approximately 16 times faster than the average, but there was no apparent relationship between clearance and time to revision. Larger McKee-Farrar balls had less volumetric wear, on average, than smaller balls, and the Müller balls had the greatest wear, which may have been due to contact with the edges of recesses machined into the bearing zones of the Müller cups.

Biomechanical Phenomena↗
PubMed1996

Protein polymorphism generated by differential RNA editing of a plant mitochondrial rps12 gene.

The rps12 gene transcripts encoding mitochondrial ribosomal protein S12 are partially edited in petunia mitochondria. Different petunia lines were found vary in the extent of rps12 transcript editing. To test whether multiple forms of RPS12 proteins are produced in petunia mitochondria as a result of partial editing, we probed mitochondrial proteins with specific antibodies against edited and unedited forms of a 13-amino-acid RPS12 peptide spanning two amino acids affected by RNA editing. Both antibodies reacted with mitochondrial proteins at the expected size for RPS12 proteins. The amounts of unedited RPS12 protein in different petunia lines correlate with the abundance of unedited transcripts in these plants. Unedited rps12 translation products are also detected in other plant species, indicating that polymorphism in mitochondrial rps12 expression is widespread. Moreover, we show that RPS12 proteins recognized by both edited-specific and unedited-specific antibodies are present in a petunia mitochondrial ribosome fraction. These results demonstrate that partially edited transcripts can be translated and that the protein product can accumulate to detectable levels. Therefore, genes exhibiting incompletely edited transcripts can encode more than one gene product in plant mitochondria.

Amino Acid Sequence↗
PubMed1996

Androgen-regulated proliferation and gene transcription in a prostate smooth muscle cell line (PS-1).

Androgens play a key role in directing stromal-epithelial interactions in prostate gland development, in maintenance of adult phenotype, and in disease progression. To address the molecular mechanisms of androgen action in prostate stromal cells and the genes regulated by androgens in this cell type, a stromal cell line (PS-1) was developed from rat ventral prostate. The PS-1 cell line was adapted to chemically defined media and characterized as smooth muscle based on expression of desmin, smooth muscle alpha-actin, and nuclear androgen receptor, markers for prostate smooth muscle. To examine steroid hormone regulation, PS-1 cells were analyzed for response to a panel of steroid hormones in serum-free, chemically defined media. PS-1 cells were significantly growth stimulated by physiological concentrations of androgens (10nM) relative to other steroids tested. To ascertain whether this cell line could be used to examine androgen-regulated gene expression, differential display PCR was used to demonstrate the presence of androgen-regulated transcripts and provide the initial steps in cloning these genes. Replicate differential display PCR analyses showed consistent androgen stimulation of 16 messenger RNA species. Northern analysis confirmed androgen regulation of 6 species. Sequence analysis of each indicated no regions of significant homology or direct matches to existing sequences. Further study of the PS-1 stromal cell line and androgen-regulated genes identified here will provide a novel in vitro system for defining molecular mechanisms of androgen action in prostate stromal cells and the significance of stromal androgen-regulated genes to stromal-epithelial interactions.

Androgens↗
PubMed1996

[Calculation of specific evolution rate of proteins based on phylogenetic].

A method of calculating specific protein evolution distance and evolution rate is presented. This is based on the reconstruction of the phylogenetic tree and on the deduction of ancestral sequences according to temporally homologous protein sequences. The evolution distance and evolution rate are calculated from the difference percentage of the sequence between the temporal and deduced ancestral sequences. A comparison with Dayoff simulation method and an example of using it to calculate the specific evolution rates of mammalian erythropoietin. The method presented here would be specially useful under certain circumstances.

Amino Acid Sequence↗
PubMed1996

A model for keratinocyte gene therapy: preclinical and therapeutic considerations.

Gene transfer to the skin is essential for correcting genetic disorders and studying skin biology. Previous attempts at in vivo gene transfer have employed replication-deficient adenovirus injected subcutaneously and plasmid DNA propelled by a gene gun. In this report, we used mouse skin as a model and evaluated the efficiency of these two methods. Using the luciferase reporter gene, we found that adenovirus injected subcutaneously transduced primarily cells in the dermis. However, particle bombardment of skin by gene gun delivered the reporter gene mainly into the epidermis. When mouse skin was bombarded with a DNA construct expressing human TGF-alpha, the epidermis of the treated mice showed localized epidermal acanthosis and hypergranulosis, which resembled the histological phenotype of previously described transgenic mice overexpressing TGF-alpha in the epidermis. These results suggest that the gene gun may be an effective tool for epidermal gene transfer and could be potentially useful in determining in vivo effects of growth factors and cytokines on the epidermis.

Adenoviruses, Human↗
PubMed1996

[Studies on gelatin microsphere loaded ligustrazine hydrochloride for lung targeting].

The effect of ligustrazine hydrochloride (LTH) on depressing pulmonary artery hypertension has been proved in recent studies. In an attempt to prepare a lung targeting dosage form, ligustrazine hydrochloride gelatin microspheres (LTH-GMS) were prepared by the method of emulsion process, using 2:1 as the weight ratio of LTH to gelatin. The preparation technique was optimized and the appearance, particle size and size distribution, LTH content, in vitro release, stability, and in vivo distribution of LTH-GMS were studied. The results showed that the mean diameter of LTH-GMS measured by Coulter counter was 12.65 microns, 87.5% of the microspheres ranging from 5 to 24.9 microns. The average content of LTH in LTH-GMS was 16.49% +/- 0.49% (n = 3) with an average extent of entrapment of about 89% and the LTH-GMS were stable for three months when stored in refrigerator or at room temperature. The release profile in vitro (pH 7.8-8.0 phosphate buffer) can be described by first-order kinetic equation. The release t1/2 of LTH when given LTH-GMS was about 6 times as much as that of original LTH. The relative distribution percentage of LTH-GMS in lung when determined 20 min after iv administration to mice was significantly higher than those of LTH-GMS in other tissues and blood, and was about 6 times as much as that of the LTH solution control group in lung under the same conditions.

Animals↗
PubMed1996

[Study on the principles of base change in 4-fold degenerate sites of protein coding genes].

The relationship of transition and transversion of base change in 4-fold degenerate sites of coding genes of 6 proteins, which showed 70% homology in wmposition investigated. Taking into account of the effects of base composition on base substitution, it was found that the transition-transversion bias was evident but not as pronounced as in mitochondrial DNA. Comparison of different kinds of transition or transversion indicated that they happened with rather equal vate, with 0.65-0.73 for different kinds of transversion and 0.99 for A-G transition assuming that T-C transition was 1. The causes for transition-transversion bias is discussed and it is proposed that mutagens may play a role in it.

Animals↗
PubMed1995

Purification of a novel protein (ps20) from urogenital sinus mesenchymal cells with growth inhibitory properties in vitro.

Our previous studies have characterized mesenchyme-derived proteins to identify biologically active proteins and novel markers for stromal cell paracrine action relative to stromal-epithelial interactions. Previous reports have characterized properties of a growth inhibitory activity (to bladder and prostatic epithelial cells), secreted by U4F fetal rat urogenital sinus mesenchymal cells, not cross-reactive with antibodies to known cytokines, and provisionally termed UGIF. The present study reports the characterization, purification, and biological properties of a 20-21-kDa protein responsible for UGIF activity. The 20-21-kDa protein (termed ps20) was purified to near homogeneity, the amino-terminal sequence was determined, and biological properties were characterized in vitro. Amino-terminal sequence analysis indicated no direct matches or regions of homology with known proteins. Purified ps20 induced a linear and saturable inhibition of [3H]thymidine incorporation in PC-3 prostatic carcinoma cells (half-maximal activity at 2.6 nM), inhibited cell proliferation (increased population doubling time from 19.8 to 25.8 h), and induced a 210% stimulation in the synthesis of secreted proteins. These data suggest that ps20 may be a candidate paracrine effector protein and may play a role in stromal-epithelial cell interaction in the prostate gland.

Animals↗
PubMed1995

Molecular cloning and functional characterization of the Xenopus Ca(2+)-binding protein frequenin.

Frequenin was originally identified in Drosophila melanogaster as a Ca(2+)-binding protein facilitating transmitter release at the neuromuscular junction. We have cloned the Xenopus frequenin (Xfreq) by PCR using degenerate primers combined with low-stringency hybridization. The deduced protein has 70% identity with Drosophila frequenin and about 38-58% identity with other Ca(2+)-binding proteins. The most prominent features are the four EF-hands, Ca(2+)-binding motifs. Xfreq mRNA is abundant in the brain and virtually nondetectable from adult muscle. Western blot analysis indicated that Xfreq is highly concentrated in the adult brain and is absent from nonneural tissues such as heart and kidney. During development, the expression of the protein correlated well with the maturation of neuromuscular synapses. To determine the function of Xfreq at the developing neuromuscular junction, the recombinant protein was introduced into Xenopus embryonic spinal neurons by early blastomere injection. Synapses made by spinal neurons containing exogenous Xfreq exhibited a much higher synaptic efficacy. These results provide direct evidence that frequenin enhances transmitter release at the vertebrate neuromuscular synapse and suggest its potential role in synaptic development and plasticity.

Amino Acid Sequence↗
PubMed1995

Probing the "message:address" sites for chemoattractant binding to the C5a receptor. Mutagenesis of hydrophilic and proline residues within the transmembrane segments.

The C5a anaphylatoxin ligand-receptor interaction on polymorphonuclear granulocytes stimulates chemotaxis, degranulation, and the oxidative burst. The receptor is a member of the large G-protein-coupled family. The ligand is a cationic peptide of 72 amino acids derived from the C5 component of complement and has been shown to have a number of structural requirements for interaction with the receptor. In order to probe the potential interaction sites between ligand and receptor, we constructed a series of mutated receptor molecules, targeting cysteines, prolines, and additional amino acids of interest because of combinations of charge or hydrophobicity and putative location with respect to the membrane. Transfected mutant receptors were analyzed for cell surface expression, ligand binding, and ligand-activated phospholipase C activity. The receptors created can be placed generally in four distinct classes: those which bind and signal like the natural receptor; those which bind but fail to transduce signals; those which are expressed but neither bind nor transduce signal; and those which are not expressed at the cell surface.

Amino Acid Sequence↗
PubMed1995

Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus.

In Drosophila the amount of neurogenic ectoderm, from which the central nervous system (CNS) derives, is regulated by a dorsal-ventral system of positional information in which two secreted molecules of antagonistic functions, decapentaplegic (dpp) and short-gastrulation (sog), play fundamental roles. The vertebrate homologue of dpp is either bmp-4 or bmp-2 (ref. 5), and the homologue os sog is chd (s-chordin). In Xenopus the CNS is induced by signals emanating from the organizer, and two proteins secreted by the organizer, noggin and follistatin, have been shown to induce neural tissue in animal-cap assays. Here we report that Chd, another organizer-specific secreted factor, has neuralizing activity and that this activity can be antagonized by Bmp-4. Inhibition of the function of the endogenous Bmp-4 present in the animal cap also leads to neural differentiation. We suggest that conserved molecular mechanisms involving chd/sog and bmp-4/dpp gene products pattern the ectoderm in Xenopus and in Drosophila.

Animals↗
PubMed1995

A conserved system for dorsal-ventral patterning in insects and vertebrates involving sog and chordin.

Dorsal-ventral patterning within the ectoderm of the Drosophila embryo requires seven zygotic genes, including short gastrulation (sog). Here we demonstrate that sog, which is expressed in the ventrolateral region of the embryo that gives rise to the nerve cord, is functionally homologous to the chordin gene of Xenopus, which is expressed in the dorsal blastopore lip of the embryo and in dorsal mesoderm, in particular the notochord. We show by injections of messenger RNA that both sog and chordin can promote ventral development in Drosophila, and that sog, like chordin, can promote dorsal development in Xenopus. In Drosophila, sog antagonizes the dorsalizing effects of decapentaplegic (dpp), a member of the transforming growth factor-beta family. One of the dpp homologues in vertebrates, bmp-4, is expressed ventrally in Xenopus and promotes ventral development. We show that dpp can promote ventral fates in Xenopus, and that injection of sog mRNA counteracts the ventralizing effects of dpp. These results suggest the molecular conservation of dorsoventral patterning mechanisms during evolution.

Amino Acid Sequence↗
PubMed1995

Neutral endopeptidase modulation of septic shock.

Neutral endopeptidase (NEP; EC. 3.4.24.11) is a type 2 cell surface metalloprotease known by a variety of eponyms, including enkephalinase, common acute lymphoblastic leukemia antigen, and CD10. Identified substrates are largely neural or humoral oligopeptide agonists, and the enzyme functions to terminate signaling by degrading the ligand, analogously to acetylcholine/acetylcholinesterase. Targeted disruption of the NEP locus in mice results in enhanced lethality to endotoxin shock with a pronounced gene dosage effect. The site(s) of action appears downstream from release of tumor necrosis factor and interleukin-1 since NEP-deficient animals demonstrate increased sensitivity to these mediators as well. This unexpected finding indicates an important protective role for NEP in septic shock.

Animals↗
PubMed1995

Nitric oxide mediates activity-dependent synaptic suppression at developing neuromuscular synapses.

Temporal correlation between pre- and postsynaptic activities is an important mechanism that regulates synaptic connectivity during development and synaptic plasticity in the adult. In developing neuromuscular junctions, postsynaptic activity is critical in functional suppression and, ultimately, elimination of the synapses. Although repetitive postsynaptic firing asynchronous to the presynaptic activity results in a persistent synaptic suppression, the underlying molecular mechanism remains unknown. Here we provide evidence that nitric oxide (NO), a free radical implicated in several forms of synaptic plasticity, may serve as a retrograde signal for activity-dependent suppression in the neuromuscular synapse. NO donors and activators of the cyclic GMP pathway suppressed spontaneous and evoked synaptic currents. Moreover, the synaptic suppression induced by repetitive postsynaptic depolarization was prevented by the NO-binding protein haemoglobin and by inhibitors of NO synthase. Thus, synaptic suppression may be triggered by NO released from a postsynaptic myocyte that fires asynchronously to the presynaptic terminal.

Action Potentials↗