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Biomedical subjects

B Hu

Publications and source records attributed to B Hu.

At least 253 records · Page 14Linked to original sources
PubMed1993

High level expression of human prourokinase cDNA in Chinese hamster ovary cells.

We have used Chinese hamster ovary (CHO) cells to express high levels of human prourokinase gene cDNA with recourse to construction of good expression vector, the improvement of transfection technique and gene coamplification. First, we constructed expression plasmid pMG10102 by placing pro-UK cDNA under the control of SR alpha promoter/SV40 polyadenylation signals and expressed it transiently in COS-7 cells. Expression level was about 5 times higher than with SV40 early promoter. Linear plasmids pMG10102 and pSV2-dhfr were then cotransfected into CHO-dhfr cells by calcium phosphorate coprecipitation and cells were cultured in selective medium. Twenty transformants expressing pro-UK were picked, the range of expression levels was 12.5-100IU/10(6) cells/day. When subjected to stepwise selection of methotrexate (MTX), the stable cell lines were obtained that secreted up to 400-500IU/10(6) cells/day. Western blot analysis showed that molecular weight of secreted recombinant pro-UK was the same as that of natural pro-UK, which is 52 kDa, and more than 60% of expression production was single chain urokinase (rscUK) without protease inhibitor in medium.

Animals↗
PubMed1993

Cloning of human prourokinase cDNA without the signal peptide and expression in Escherichia coli.

Human prourokinase (pro-UK) cDNA without the signal peptide was obtained using synthetic oligonucleotide and DNA recombination techniques and was successfully expressed in E. coli. The plasmid pMMUK which contained pro-UK cDNA (including both the entire coding sequence and the sequence for signal peptide) was digested with Hind III and PstI, so that the N-terminal 371-bp fragment could be recovered. A 304-bp fragment was collected from the 371-bp fragment after partial digestion with Fnu4HI in order to remove the signal peptide sequence. An intermediate plasmid was formed after this 304-bp fragment and the synthetic oligonucleotide was ligated with pUC18. Correctness of the ligation was confirmed by enzyme digestion and sequencing. By joining the PstI-PstI fragment of pro-UK to the plasmid we obtained the final plasmid which contained the entire coding sequence of pro-UK without the signal peptide. The coding sequence with correct orientation was inserted into pBV220 under the control of the temperature-induced promoter PRPL, and mature pro-UK was expressed in E. coli at 42 degrees C. Both sonicated supernatant and inclusion bodies of the bacterial host JM101 showed positive results by ELISA and FAPA assays. After renaturation, the biological activity of the expressed product was increased from 500-1000IU/L to about 60,000IU/L. The bacterial pro-UK showed a molecular weight of about 47,000 daltons by Western blot analysis. It can be completely inhibited by UK antiserum but not by t-PA antiserum nor by normal rabbit serum.

Base Sequence↗
PubMed1993

Effect of cholesterol oxides on prostacyclin production and platelet adhesion.

Prostacyclin (PGI2) is synthesized primarily by endothelial cells, is essential for maintenance of vascular integrity, and may play a role in atherogenesis. Human umbilical vein endothelial cells in culture were incubated with either pure cholesterol, 25-hydroxycholesterol, 7-ketocholesterol, cholesterol 5 alpha,6 alpha-epoxide or cholestane-3 beta,5 alpha,6 beta-triol at 10 micrograms/ml culture medium concentration for 12 hours and 24 hours. PGI2 production measured by radioimmunoassay of 6-keto PGF1 alpha, the stable metabolite of PGI2 was inhibited by 39.6%, 27.3%, 40.1% and 31.9% after incubation with 25-hydroxycholesterol, 7-ketocholesterol, cholesterol 5 alpha, 6 alpha-epoxide or cholestane-3 beta,5 alpha,6 beta-triol for 12 hours respectively. Further inhibitory effects were shown after 24 hours of incubation with 25-hydroxycholesterol and 7-ketocholesterol. Platelet adhesion onto endothelial cell monolayers measured by 111In-labeled platelets was enhanced by 104%, 54% and 37% after incubation with cholestane-3 beta, 5 alpha,6 beta-triol, 25-hydroxycholesterol, and 7-ketocholesterol at 10 micrograms/ml concentration for 12 hours respectively. Pure cholesterol at the same concentration had no effect on PGI2 production or platelet adhesion.

Arteriosclerosis↗
PubMed1992

Immune complexes that bind to ELISA plates not coated with antigen in mice infected with lactate dehydrogenase-elevating virus: relationship to IgG2a- and IgG2b-specific polyclonal activation of B cells.

We have further investigated the nature of IgG-containing complexes of 150-300 kD that rapidly appear in the circulation of mice of various strains after infection with lactate dehydrogenase-elevating virus (LDV) and are recognized and quantitated by their binding in the presence of 0.05% Tween 20 to certain enzyme-linked immunosorbent assay (ELISA) plates with high protein affinity that have not been coated with protein antigen (5). These binding complexes have been found to contain primarily IgG2a or, in some mice, IgG2b. Their isotype specificity and time course of formation correlated with those of the polyclonal production of immunoglobulins in these mice, as measured by increases in total IgG2a or IgG2b in the circulation. In contrast, anti-LDV antibodies exhibited much broader isotype specificities in all mouse strains investigated. Depletion of BALB/c mice of CD4+T cells or lack of T cells in nude Swiss mice only partly reduced the polyclonal activation of B cells and the formation of ELISA plate-binding complexes, whereas anti-LDV antibody formation was completely blocked. Only a small proportion of the total IgG2a or IgG2b formed as a result of the LDV-induced polyclonal activation of B cells was recovered in plate-binding complexes, which sedimented in sucrose density gradients between 150 and 300 kD. Diverse monoclonal antibodies of different IgG isotopes did not bind to the plates at concentrations at which LDV-induced immune complexes exhibited binding activity. We suggest that the LDV-induced immune complexes do not contain anti-LDV antibodies, but are complexes of auto-antibodies and self-antigen(s). However, additional features must be responsible for the high affinity of these complexes for ELISA plates since various immune complexes formed in vitro failed to bind to the plates, and binding activity of the immune complexes formed in LDV-infected mice could not be regenerated in vitro once the complexes had been dissociated by a low pH treatment.

Animals↗
PubMed1992

Correlation between levels of immunoglobulins and immune complexes in plasma of C57BL/6 and C57L/J mice infected with MAIDS retrovirus.

Infection with helper-free, defective MAIDS murine leukemia virus (MuLV) caused a rapid polyclonal activation of B cells in 0.75-, 2-, and 6-month-old C57L/J mice (H-2b, Fv-1n/n), similar to that in C57BL/6 mice (H-2b, Fv-1b/b), which was recognized by elevated plasma immunoglobulin concentrations. However, changes in plasma immunoglobulin levels differed in C57BL/J and C57BL/6 mice. In C57L/J mice, infection resulted in a rapid increase in plasma IgM and IgG2a, and the elevation of IgG2a persisted undiminished for 21 weeks. Levels of IgG2b also became slightly elevated, but those of IgG1 and IgG3 were not significantly affected. Plasma of 6 to 7-month-old C57BL/6 mice contained already high levels of IgM (30-40 mg/ml), which persisted undiminished in uninfected mice but decreased progressively in infected mice to 10% of the original concentration during 25 weeks of observation. In C57BL/6 mice, plasma IgG1 and IgG2b as well as IgG2a became similarly elevated after infection but also only transiently. Their levels began to decrease progressively about 10 weeks after infection and fell to far below the maximum concentration observed. The drastic loss of plasma IgM and IgGs observed in C57BL/6 mice during the later stages of MAIDS MuLV infection did not seem to be a consequence of the polyclonal activation of B cells per se but seemed to reflect additional immunological abnormalities arising in infected C57BL/6 but not C57L/J mice. In both mouse strains these changes in plasma Ig levels correlated with the formation of Ig-containing immune complexes that bound to high-affinity, protein-binding ELISA plates in the absence of antigen coating, which may represent unusual forms of self-antigen-antibody complexes.

Animals↗
PubMed1992

Rat supraoptic neurons are resistant to glutamate neurotoxicity.

Magnocellular neurosecretory cells of the supraoptic nucleus (SON) are thought to be endogenously resistant to glutamate toxicity. In this study, we sought physiological and morphological evidence of this resistance in rats that received multiple peri-nuclear injections of ibotenate. In this preparation, ibotenate produced a large necrotic zone encompassing the SON but not within the nucleus itself. Extracellular recordings in vivo from 68 'spared' SON neurons from lesioned rats revealed normal patterns of electrical activity. Intracellular analysis in vitro from 13 'spared' SON neurons indicated that their intrinsic membrane properties, osmosensitivity and spontaneous synaptic activity did not differ significantly from that of controls. We conclude that SON neurons retain both a morphological and a physiological resistance to glutamate neurotoxicity.

Animals↗
PubMed1992

NMDA receptor-mediated rhythmic bursting activity in rat supraoptic nucleus neurones in vitro.

1. Intracellular recordings were obtained from 112 supraoptic nucleus magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus maintained in vitro. The effects of glutamate receptor agonists and antagonists were examined at 32-34 degrees C. 2. In control solutions, spontaneously active (> 5 Hz) phasic or continuous neurones showed interspike interval distributions slightly skewed toward short intervals, but did not feature pauses in the 0.4-2 s range. Current injection to alter the rate of cell discharge shifted the histograms according to the mean firing rate, but failed to induce intermittent pauses in the 0.4-2 s range. 3. Application of N-methyl-D-aspartate (NMDA) induced a mode of firing in which bimodal interspike interval distributions reflected a high incidence of clusters of short interspike intervals (0.5-1.5 s) recurring every 1-3 s. In contrast, firing evoked by application of D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) was not associated with a clustering of impulse discharge. 4. The putative endogenous excitatory amino acid transmitters L-glutamate, L-aspartate and quinolinate all mimicked the effects of NMDA. Clustered spiking responses to these agents were reversibly blocked by D,L-2-amino-5-phosphono-valerate (APV), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, the non-NMDA receptor ligands kainate and quisqualate caused CNQX-sensitive increases in firing rate, but these responses were not associated with the appearance of clustered activity. 5. When applied to cells showing negative resting potentials (< -70 mV), or to neurones hyperpolarized by current injection, responses to NMDA consisted of rhythmic (approximately 1 Hz) voltage oscillations associated with bursts of spike discharge. In the presence of TTX, NMDA could induce subthreshold voltage oscillations in the absence of action potentials. 6. Application of a voltage clamp to potentials between -75 and -55 mV during rhythmic bursting responses failed to reveal any rhythmic oscillation of the membrane current. In all cases, rhythmic bursting activity resumed upon returning to the current-clamp mode. 7. Rhythmic bursting responses to NMDA application were abolished in Mg(2+)-free solutions, suggesting that the voltage dependence of NMDA channels served to promote regenerative voltage changes throughout the cycle. The NMDA-induced current itself, however, did not appear to decrease with time, suggesting that a distinct, outward current, was necessary to initiate the repolarizing phase of each cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1992

[Synthesis and radiosensitizing activity of benzimidazoles].

In search for effective radiosensitizer with low neurotoxicity, benzimidazole compounds were synthesized. Reaction of 2-nitrobenzimidazole with ethyl chloroformate yielded ethyl alpha-(2-nitrobenzimidazolyl-1)-formate(1) or ethyl alpha-(2-hydroxybenzimidazolyl-1)-formate(2), depending upon the solvents used. Reaction of 2-nitrobenzimidazole with 1,2-epoxy-3-chloropropane gave a cyclized compound which was confirmed to be benzimidazo(1,2b)-5'-chloromethyl-oxazolidine(3). In attempt to increase hydrophilicity, 1-substituted 2(3'-pyridyl)-5-nitrobenzimidazoles were prepared by reaction of 2-(3'-pyridyl)-5(6)-nitrobenzimidazole(4) with alkyl epoxides or ethyl chloroacetate. Some of the compounds synthesized were tested for radiosensitizing activity in Ehrlich ascites carcinoma-bearing mice. Preliminary results showed that some compounds have radiosensitizing activity. The radiosensitizing enhancement ratio (SER) of compounds 3, 5 and 6 were found to be 1.50, 1.52 and 1.65 respectively.

Animals↗
PubMed1992

[The experimental studies of the effect of Forskolin on the lowering of intraocular pressure].

The effect of the domestic Forskolin on lowering the intraocular pressure (IOP) of rabbits was studied. The results showed that the Forskolin significantly lowered the normal IOP of rabbits and blocked the ocular hypertension induced by water load in rabbits (p < 0.01). The maximum decrease value of 2%, 1% and 0.5% of the Forskolin was 0.59. 0.36 and 0.19 kPa (1 kPa = 7.5 mmHg), which showed the noticeable dose-effect relationship. Topical ocular application of Forskolin lowered IOP in 1/2 hour, reached to a peak in 2-3 hours and remained significantly for 10 hours. The pupillary diameter did not change when IOP were reduced. Furthermore, the Forskolin had potent stimulative properties to adenylate cyclase (AC). The greater the ability of the Forskolin to stimulate AC, the stronger the effect of IOP lowering.

Adenylyl Cyclases↗
PubMed1992

[Synthesis and radiosensitizing activity of nitro-arylimino-diethyl-sodium thiosulfate and nitro phenylalanine derivatives].

A series of compounds was synthesized, these compounds were tested for Hela-S3 cells in vitro for radiosensitizing activity. Five of them are 2,2'-(arylimino)-diethyl-sodium thiosulfate and two of them are phenylalanine derivatives. Most of them showed various degrees of radiosensitizing activity. Among them, SER of L07 was 1.89 at 3 mmol, and had low cytotoxicity to Hela-S3 cells, ID50 was 18.8 mmol. The relationship between radiosensitizing effects and chemical structure was discussed. It offers a base for further exploration of selectively hypoxic cell radiosensitizers.

HeLa Cells↗
PubMed1991

Phorbol esters and cyclic AMP activate AMP deaminase in adult rat cardiac myocytes.

Using rapid deenergization as a probe for adenylate deaminase activity in intact adult rat cardiac myocytes, we have previously established that IMP formation is enhanced by alpha-adrenergic agonists. In the present study, the effect of adrenergic agents on adenylate deaminase was further characterized. Phenylephrine (PE)3 increased IMP production in a dose-dependent fashion with an EC50 of 8 x 10(-7) M. The response to PE was reversed within 10 min by the alpha 1-antagonist, prazosin. Likewise, adenylate deaminase was also activated in ventricular myocytes challenged with phorbol 12-myristate 13-acetate (PMA, EC50 = 5 nM); cardiac cells presented with 100 nM PMA increased IMP production from 4.4 +/- 0.5 (control) to 15.7 +/- 0.9 nmol/mg protein when subsequently deenergized. The effects of PMA and PE were attenuated 85 +/- 5% and 96 +/- 4%, respectively, by pretreatment of cells with 150 nM staurosporine, an inhibitor of protein kinase C. Furthermore, incubation of cardiac cells with 1 microM PMA for 24 h blunted the response to both PMA and phenylephrine 85-90%. Elevating cyclic AMP (cAMP) content to greater than 15 pmol/mg by treatment with forskolin or isoproterenol plus isobutylmethylxanthine also resulted in enhanced adenylate deaminase activity, but this stimulatory effect was not abolished by 24 h incubation with 5 microM PMA. Forskolin and PMA-induced increases in IMP production appeared to be additive. However, 0.5 microM isoproterenol inhibited the cellular response to phenylephrine by about 30% but did not affect PMA-stimulated adenylate deaminase activity. We conclude that both cAMP and protein kinase C stimulate adenylate deaminase, perhaps through selective activation of different isoforms. However, cAMP also exerts partial inhibition on alpha-adrenoreceptor-mediated increases in IMP production.

AMP Deaminase↗
PubMed1991

Angiotoxicity and atherogenicity of cholesterol oxides.

Cholesterol in the diet can readily autoxidize and be absorbed and transported in plasma lipoproteins. Cholesterol oxides can also be endogenously produced in tissues via free-radical-induced reactions. Some cholesterol oxides, notably cholestane-3 beta, 5 alpha, 6 beta-triol and 25-hydroxycholesterol, have been shown to cause injury to vascular endothelial and smooth muscle cells, to alter LDL receptor function, to enhance cholesteryl ester accumulation, to inhibit prostacyclin production, and to induce experimental atherosclerosis alone or in combination with cholesterol. An epidemiological study examining relationships between atherosclerosis and plasma levels of cholesterol oxides as independent risk factors may provide additional insights regarding the roles of cholesterol oxides in atherogenesis.

Animals↗