Biomedical subjects

B Lu

Publications and source records attributed to B Lu.

At least 217 records · Page 12Linked to original sources

PubMed1995

Beta-2 microglobulin is mitogenic to PC-3 prostatic carcinoma cells and antagonistic to transforming growth factor beta 1 action.

Previous studies have identified a M(r) 12,000 protein in rat prostatic stromal cell-conditioned medium with growth stimulatory activity to human prostatic carcinoma cells as a direct match with beta 2-microglobulin (beta 2-m). The present study was conducted to characterize the activities of human beta 2-m directly, using commercially available, purified human beta 2-m. Beta 2-m was assayed for growth stimulatory activity to human PC-3 prostatic carcinoma cells and rat PS-1 prostatic stromal cells and for antagonistic activity to transforming growth factor beta 1 (TGF-beta 1)-induced growth inhibitory actions. Beta 2-m acted to stimulate [3H]thymidine incorporation in PC-3 cells in a linear, concentration-dependent and saturable manner in serum-free medium. Beta 2-m stimulated cell proliferation and significantly decreased population doubling times in both PC-3 and PS-1 cell lines. At half-maximal concentrations of TGF-beta 1 and lower, beta 2-m acted in a concentration-dependent, antagonistic manner, acting to stimulate growth-inhibited PC-3 cells to fully neutralize TGF-B1 activity. In contrast, cells exposed to maximum activity TGF-beta 1 concentrations were refractory to beta 2-m action, regardless of the concentration tested. This represents the first report to demonstrate a growth-stimulatory activity of B2-m with carcinoma/epithelial cells and to show beta 2-m antagonistic activity to TGF-B1 growth-induced inhibition. Beta 2-m has been shown previously to associate with hormone/growth factor receptors. Together, these data suggest that beta 2-m may play a role in modulating cell proliferation, possibly through modification of ligand/receptor kinetics. Owing to the elevation of both beta 2-m and TGF-beta 1 in many dysplastic-neoplastic conditions, beta 2-m may be relevant to mechanisms of abnormal proliferation disorders and in modulating TGF-beta 1 mechanisms of actions.

Cell Division↗

PubMed1995

Accelerated structural maturation induced by synapsin I at developing neuromuscular synapses of Xenopus laevis.

The role of synapsin I, a synaptic vesicle-associated phosphoprotein, in the maturation of nerve-muscle synapses was investigated in nerve-muscle co-cultures prepared from Xenopus embryos loaded with the protein by the early blastomere injection method. The stage of maturation of the synapses was analysed by electron microscopy as well as by whole-cell patch-clamp recording. The acceleration in the functional maturation of neuromuscular synapses induced by synapsin I was accompanied by a profound rearrangement in the ultrastructure of the nerve terminal. Nerve terminals formed by synapsin I-loaded neurons were characterized by a higher number of small synaptic vesicles organized in clusters and predominantly localized close to the nerve terminal plasma membrane, a smaller number of large dense-core vesicles and no significant change in the number of coated vesicles. Precocious development of active zone-like structures as well as deposition of basal lamina into the synaptic cleft were also observed at these synapses. These results support a role for synapsin I in the architectural changes which occur during synaptogenesis and lead to the maturation of quantal neurotransmitter release mechanisms.

Animals↗

PubMed1995

Herpes simplex virus type 1 amplicon vectors with glucocorticoid-inducible gene expression.

A glucocorticoid-inducible transcription unit, composed of reiterated steroid-responsive cis elements, a synthetic promoter, and the human growth hormone gene (hGH), was inserted into herpes simplex virus type 1 (HSV-1) amplicon vectors to determine if regulated expression can be achieved in cell lines and primary hepatocytes. Initial constructs were prepared in a HSV amplicon vector that contained a 0.96-kb DNA fragment encompassing an origin of DNA replication of the short repeat (ORIs) of HSV-1, similar to ORIs-containing fragments used in many of the existing HSV amplicon vectors. In the absence of glucocorticoids, these constructs produced a high basal level of hGH expression in cells both transfected with naked amplicon DNA and infected with packaged amplicon virus. To lower basal expression, the larger ORIs was replaced with a 237-bp ORIs DNA fragment that was devoid of the many trans-activation elements flanking the larger ORIs, but that could still support DNA replication of amplicon vectors. HSV amplicon vectors with this smaller ORIs produced up to 30-fold hGH induction upon glucocorticoid treatment in virally transduced 293 cells, and up to 50-fold induction in transduced primary rat hepatocytes. The kinetics of hGH accumulation after induction and the dependence of hGH expression with respect to dexamethasone concentration were characterized in both cell types. These vectors have application for experiments in which highly efficient gene transfer and inducible gene expression are required.

Base Sequence↗

PubMed1995

Regulation of postsynaptic responses by calcitonin gene related peptide and ATP at developing neuromuscular junctions.

Neuronal factors co-released with neurotransmitters may play an important role in synapse development and function. Calcitonin gene related peptide (CGRP) and adenosine 5'-triphosphate (ATP), two principal neuromodulators present in the motor nerve terminals, were studied for their roles and mechanisms during early development of neuromuscular synapses in Xenopus nerve--muscle co-cultures. CGRP treatment increased the decay time and amplitude of spontaneous synaptic currents (SSCs) recorded from innervated myocytes, without affecting SSC frequency, suggesting a postsynaptic mechanism. ATP also increased the SSC amplitude and decay time. In addition, ATP was shown to potentiate the responses of isolated myocytes to iontophoretically applied acetylcholine (ACh). Single-channel recording from isolate myocytes showed that both CGRP and ATP specifically increased the open time of embryonic-type, low-conductance ACh channels. Pharmacological experiments suggest that the CGRP actions were mediated by cAMP-dependent protein kinase (PKA), while ATP exerted its effects by binding to P2 purinoceptors and thereby activating protein kinase C (PKC). Moreover, the effects of CGRP and ATP on ACh channel activity were restricted to immature myocytes. Taken together, these results suggest that endogenous CGRP and ATP co-released with ACh from the nerve terminal may promote synaptic development by potentiating postsynaptic ACh channel activity during the early phase of synaptogenesis.

Adenosine Triphosphate↗

PubMed1995

[Studies on lung targeting gelatin microspheres of mitoxantrone].

Lung targeting gelatin microspheres of mitoxantrone (DHAQ) were prepared by a two-step method. The diameter of 87.36% of the DHAQ gelatin microspheres (DHAQ-GMS) was in the range of 5.1 to 25.0 microns. Release of the drug from the DHAQ-GMS in vitro became much slower and its t1/2 was 4 times longer than that of pure DHAQ. The characteristic peak of heat absorption on the differential thermal analysis curve was at 133 degrees C and almost no change was observed after the DHAQ-GMS were stored for 3 months at 37 degrees C (relative humidity 75%). The distribution test in vivo in mice indicated that the lung targeting effect of the DHAQ-GMS was obvious and that the targeting efficiency of the lung compared to other organs and blood increased 3 to 35 times. Kinetic behavior of the drug in mouse lung could be described by one open compartment model, and the average residual time increased by 10 h.

Animals↗

PubMed1995

[Study on the lung targeting gelatin microspheres of streptomycin sulphate].

This paper is reported the technology of lung targeting gelatin microspheres of streptomycin sulphate (SMS). The microspheres were prepared with natural biodegradable gelatin as the load material and castor oil as the oil phase. The experimental conditions were optimized, the mean volume diameter obtained being 9.7 microns and the mean rate of encapsulation 15.69%. The content, shape and size of the microspheres showed no remarkable change after storage at 37 degrees C RH 75% for 3 months. Activation energy of heat decomposition E = 75.86kJ/mol. In vitro, the SMS release rate was found to accord with Higuchi equation with t1/2 = 8.6h. In vivo (rabbits) the gelatin microspheres were proved to be concentrated in the lung.

Animals↗

PubMed1995

Neurotrophins promote maturation of developing neuromuscular synapses.

Although the effects of neurotrophins on survival and differentiation of various neuronal populations have been well studied, little is known about their role in synaptic development and function. We have investigated the long-term effects of neurotrophins in the maturation of neuromuscular synapses in Xenopus nerve-muscle cocultures. BDNF and NT-3, but not NGF, elicited significant changes in several properties of spontaneous synaptic currents (SSCs), indicative of more mature synapses. Most synapses treated by the neurotrophins exhibited a bell-shaped distribution of SSC amplitudes, which reflects mature quantal secretion. The neurotrophins also potentiated the efficacy and reliability of stimulus-induced synaptic transmission. Moreover, BDNF and NT-3 increased the levels of the synaptic vesicle proteins, synaptophysin, and synapsin 1 in the spinal neurons. The number of varicosities per neuron also showed a significant increase after neurotrophin treatment. The effects of the neurotrophins appear to be mediated by the Trk family of receptor tyrosine kinases, primarily through a presynaptic mechanism. These results suggest that BDNF and NT-3 promote functional maturation of synapses.

Acetylcholine↗

PubMed1995

Ex vivo hepatic gene transfer in mouse using a defective herpes simplex virus-1 vector.

A defective amplicon herpes simplex virus-1 (HSV-1) vector, HSVlac, was used to transfer an E. coli lacZ reporter gene into primary hepatocytes. The lacZ gene was driven by the HSV immediate early (IE) 4/5 promoter. Use of the HSVlac vector resulted in highly efficient gene transfer. Because difficulties in culturing primary hepatocytes impose limitations in ex vivo gene therapy, we sought to determine whether use of the HSVlac vector could simplify gene transfer. Therefore, we incubated HSVlac with primary hepatocytes in suspension and found that the lacZ gene was still transferred with great rapidity and efficiency. To examine lacZ expression in transduced hepatocytes in vivo, we used a mouse hepatocyte transplantation system. In congeneic recipients of primary hepatocytes transduced with HSVlac in suspension, the lacZ gene was expressed in liver and spleen up to 2 weeks. However, survival of transplanted hepatocytes, as well as persistence of HSVlac genome in recipient organs, was demonstrated for up to an 11-week duration of the experiment. These findings suggest that in vivo regulation of the HSV IE4/5 promoter was responsible for the short-term expression of lacZ, which should be overcome by the use of liver-specific promoters. Therefore, our results indicate the feasibility of hepatic gene transfer with a defective HSV-1 vector.

Animals↗

PubMed1994

Neurotrophin 3 potentiates neuronal activity and inhibits gamma-aminobutyratergic synaptic transmission in cortical neurons.

Neurotrophins have traditionally been regarded as slowly acting signals essential for neuronal survival and differentiation. However, brain-derived neurotrophic factor and neurotrophin 3 (NT-3) have recently been reported to exert an acute potentiation of synaptic activity at the amphibian neuromuscular junction. Little is known about the role of neurotrophins on functional synapses in the central nervous system. Here we show that NT-3 rapidly increased the frequency of spontaneous action potentials, and it synchronized excitatory synaptic activities in developing cortical neurons. Moreover, the inhibitory synaptic transmission mediated by gamma-aminobutyric acid (GABA) subtype A receptors was found to be reduced by NT-3. Thus, the excitatory effects of NT-3 on spontaneous action potentials were attributable to a reduction of GABAergic transmission. Our findings, together with previous reports of rapid regulation of central nervous system neurotrophin expression by neuronal activity and of the role of GABAergic transmission in cortical plasticity, suggest a mechanism for modulation of synaptic transmission and activity-dependent synaptic modulation in cortical neurons.

Amphibians↗

PubMed1994

Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes.

A Xenopus gene whose expression can be activated by the organizer-specific homeobox genes goosecoid and Xnot2 was isolated by differential screening. The chordin gene encodes a novel protein of 941 amino acids that has a signal sequence and four Cys-rich domains. The expression of chordin starts in Spemann's organizer subsequent to that of goosecoid, and its induction by activin requires de novo protein synthesis. Microinjection of chordin mRNA induces twinned axes and can completely rescue axial development in ventralized embryos. This molecule is a potent dorsalizing factor that is expressed at the right time and in the right place to regulate cell-cell interactions in the organizing centers of head, trunk, and tail development.

Activins↗

PubMed1994

Staphylococcal protein A containing phospholipid monolayers on aqueous and solid surfaces.

The staphylococcal protein A containing monolayer is characterized on both aqueous and solid surfaces. The density of SpA in dipalmitoylphosphatidic acid (DPPA) monolayers is determined from ultraviolet spectroscopy. The influence of calcium on the SpA concentration in the monolayer is also investigated. The spectroscopic results indicate that the incorporation of SpA in the monolayer is influenced by the calcium concentration in the subphase, and more than 90% of adsorbed protein is excluded from the monolayer when calcium concentration is in the order of millimoles. The IgG binding activity of the SpA in the reconstituted membrane is influenced by the surface pressure at which the membrane is formed.

Calcium↗

PubMed1994

Characterization of modified staphylococcal protein A containing phospholipid monolayer on both solution and slide surfaces.

A method is described for incorporation of water-soluble protein Staphylococcal protein A (SpA) into phospholipid monolayer using covalent protein-lipid conjugates in detergent solution. The amphiphilic conjugates have solubility properties very similar to intergral membrane proteins. When the conjugates are applied into dipalmitoyl-phosphatidic acid monolayer, a protein containing monolayer is formed on subphase surface. The monolayer is transferred to pre-coated substrate surface to form an artificial membrane. Results show that unmodified SpA is readily ejected from the monolayer when compressing the monolayer but modified SpA incorporates into the monolayer stably. The incorporation of the protein is proportional to the lipid coupling degree. When the protein is excessively modified, the IgG binding activity of the SpA in the membrane is lost significantly.

Immunoglobulin G↗

PubMed1994

Differentiation-specific expression of human keratin 1 is mediated by a composite AP-1/steroid hormone element.

Human keratin 1 (HK1) expression is associated with the loss of mitotic activity in epidermal keratinocytes and restricted to an intermediate stage of terminal differentiation. Recently, the control elements that mediate this differentiation-specific expression were identified (Huff, C. A., Yuspa, S. H., and Rosenthal, D. (1993) J. Biol. Chem. 268, 377-384). We now report the characterization of one of these elements. Footprint analysis on a 249-base pair fragment containing the calcium responsive element (CaRE) revealed two adjacent protected regions. The 5' most footprint contains an AP-1 consensus sequence while the 3' footprint encodes two inverted repeats of the canonical hormone response recognition sequence. Deletion of the AP-1-protected region abolished the calcium response in a reporter construct. Calcium activation of the reporter construct containing the intact CaRE was unaffected by the addition of thyroid hormone or estrogen. However, vitamin D3 was able to suppress calcium induction by the CaRE, and this suppression could be abrogated by the coaddition of retinoic acid. These studies show that AP-1 factors bind to the 5' element to mediate the calcium response while members of the steroid hormone receptor superfamily interact with the 3' element to modulate the calcium response.

Base Sequence↗

PubMed1994

Functional characterization of the rat GAP-43 promoter.

GAP-43 is a highly conserved neuronal protein whose expression is spatially and temporally regulated. Because this regulation may occur, at least in part, at the level of transcription, we have begun to characterize the regions upstream of the GAP-43 transcription unit which direct its neuronal-specific expression. Functional analyses of GAP-43 promoter-reporter constructs have been performed in stably transfected cell lines, including PC12, C6 and RAT2. These data indicate that as little as 600 bp of GAP-43 5'-flanking DNA sequence directs the expression in a neuronal-specific manner. A shorter construct containing 230 bp of 5'-flanking DNA sequence defines a GAP-43 minimal promoter that is active in both neuronal and glial but not in non-neural cell lines. An upstream region, previously shown by other investigators to have promoter activity, was able to stimulate transcription when linked to the downstream minimal promoter. However, this upstream region was by itself unable to direct transcription of the reporting gene. In addition, we have demonstrated that two polypurine tracts within the 5'-flanking DNA sequence of the GAP-43 gene adopt a non-duplex configuration in plasmids, and, when studied in the context of chromosomal integration, these regions have a stimulatory effect on transcription.

Animals↗

PubMed1994

Motion observed across maxillary continuity defects stabilized with plates and screws.

The stability of fixation obtained with internal fixation devices (plates and screws) under functional loading of maxillary continuity defects was studied using fresh cadaver skulls. Micromotion (deformation) was recorded in the model across the level of the LeFort I osteotomies even when fixation was thought to be grossly stable. Deformation in this biologically static model proved to be mostly elastic in nature, and the amount of deformation in each skull appeared to be predictable from the average stability of all screws used in each plate. Such motion in viable bone could lead to permanent deformation due to resorption and remodeling of bone around the screws if initial overall fixation stability is tenuous. The results suggest that the term "rigid" is inappropriately used to describe plate and screw fixation of maxillary continuity defects.

Bone Plates↗

PubMed1994

A single homogeneous form of ATP6 protein accumulates in petunia mitochondria despite the presence of differentially edited atp6 transcripts.

Transcripts encoding ATP synthase subunit 6 (ATP6) in petunia mitochondria were shown to be edited at 15 sites, leading to 14 amino acid changes. Certain sites are partially edited, including a site that introduces a new translation termination codon that is 13 codons upstream of the genomically encoded stop codon. Transcripts lacking the new stop codon are present in an approximately 2.5:1 ratio to transcripts carrying the stop codon created by RNA editing. To investigate whether partially edited transcripts are represented as proteins, we generated an antibody against a 12-residue peptide that is specific for translation products of unedited transcripts. This antibody did not recognize any ATP6 protein in either total mitochondrial protein preparations or ATP6 samples purified by organic solvent extraction and reverse phase HPLC procedures. According to analysis by mass spectrometry, only one form of ATP6 protein accumulates in mitochondria despite the presence of abundant partially edited transcripts. Partially edited atp6 transcripts were associated with ribosomes, suggesting that a screening mechanism(s) acts cotranslationally or post-translationally to exclude the expression of incompletely edited transcripts.

Amino Acid Sequence↗

PubMed1994

Stress response of Porphyromonas gingivalis.

The heat shock response of Porphyromonas gingivalis was examined by 1- and 2-dimensional polyacrylamide gel electrophoresis after metabolic labeling with [14C]-amino acids. When P. gingivalis cells were shifted from 37 degrees C to 42 degrees C, elevated synthesis of 4 proteins with the apparent molecular weight of 92, 80, 74, and 62 kDa was observed, and synthesis of a 50-kDa protein decreased during heat shock. The 74- and 62-kDa proteins were identified as homologs of Escherichia coli DnaK and GroEL respectively by Western immunoblotting. On 2-dimensional Western blots, 2 forms of DnaK and 4 forms of GroEL were identified due to their slightly different isolelectric points. dnaK and groEL gene homologs were identified in several P. gingivalis strains and some other black-pigmented oral species. DnaK and GroEL homolog proteins were induced when P. gingivalis cells were challenged by ethanol. These proteins were not induced by oxidative stress or change in pH.

Animals↗

PubMed1993

Calcitonin gene-related peptide potentiates synaptic responses at developing neuromuscular junction.

Protein phosphorylation is important in synaptic transmission and plasticity. At the neuromuscular junction, phosphorylation of acetylcholine (ACh) receptor-channels increases the rate of agonist-induced channel desensitization. In contrast, potentiation of ACh channel activity through protein phosphorylation has not been described. We report here that calcitonin gene-related peptide (CGRP), a neuropeptide present at presynaptic motor nerve terminals, enhances the postsynaptic response at developing neuromuscular junctions by increasing the burst duration of embryonic ACh channels. The effect of CGRP on these ACh channels is mimicked by dibutyryl-cyclic AMP and by cAMP-dependent protein kinase (PKA) and prevented by a specific peptide inhibitor of PKA. Moreover, postsynaptic inhibition of PKA reduced the amplitude and decay time of spontaneous synaptic currents, suggesting that endogenous CGRP may act as a potentiating factor during the early phase of synaptogenesis.

Acetylcholine↗