Biomedical subjects

H M Huang

Publications and source records attributed to H M Huang.

At least 37 records · Page 2Linked to original sources

PubMed2000

Anti-inflammatory and neuroprotective effects of magnolol in chemical hypoxia in rat cultured cortical cells in hypoglycemic media.

Our previous studies demonstrated that magnolol protects neurons against chemical hypoxia by KCN in cortical neuron-astrocyte mixed cultures (14). In the present study, we examined whether the neuroprotective effect of magnolol involve modulating inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO), induced by KCN (hypoxia) or KCN plus lipopolysaccharide (LPS). In glucose-absent (hypoglycemia) media, KCN or KCN plus LPS induced increases in lactate dehydrogenase (LDH) activity by 32% and 34%, and PGE2 production by 12% and 32%, respectively. Both LDH and PGE2 increases were suppressed by 100 microM magnolol. In addition, although KCN or LPS alone did not increase NO generation, KCN plus LPS increased NO generation. This increase was reduced by 100 microM magnolol or 10 microM L-NAME, but the LDH increase and PGE2 production were not reduced by L-NAME. These findings suggest that the protective effects of magnolol against brain damage by KCN or KCN plus LPS in hypoglycemic media may involve inhibition of PGE2 production, but inhibition of NO generation may not be important.

Animals↗

PubMed2000

Antioxidants prevent amyloid peptide-induced apoptosis and alteration of calcium homeostasis in cultured cortical neurons.

Beta-amyloid ((A)beta) is a peptide of 39-42 amino acids that is the primary component of plaques in Alzheimer's disease (AD). The mechanism by which (A)beta expresses its neurotoxic effects may involve induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels. Cultured cortical cells were utilized to study the alterations in calcium homeostasis underlying the neurotoxic effect of (A)beta. Serum supplement B27 and vitamin E were effective in preventing neuronal death as assessed by lactate dehydrogenase (LDH) release, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and number of apoptotic nuclei. In addition, (A)beta-induced cytosolic free calcium ([Ca2+]i) was blocked by antioxidants vitamin E and U83836E, but not by N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801, or by voltage-gated calcium channel blocker nimodipine. Taken together, the results suggest that NMDA receptor and voltage-gated calcium channels are not involved in (A)beta-induced [Ca2+]i increase. This increase appeared to be the result of extracellular calcium influx by some unknown mechanisms. In addition, antioxidants such as B27 were effective in protecting cultured cortical neurons against (A)beta, and correlated with (A)beta attenuation of early calcium response.

Amyloid↗

PubMed2000

Microcalorimetric studies of interactions between proteins and hydrophobic ligands in hydrophobic interaction chromatography: effects of ligand chain length, density and the amount of bound protein.

Using isothermal titration calorimetry (ITC), this investigation directly measured the adsorption enthalpies of proteins on various hydrophobic adsorbents. Various amounts of butyl and octyl groups were attached onto CM-Sepharose to form C4 and C8, two types of hydrophobic adsorbents. The adsorption enthalpies of both trypsinogen and alpha-chymotrypsinogen A were measured at 4.0 M NaCl and pH 10.0, in which most ionic interaction was suppressed. The adsorption isotherms of both proteins on various adsorbents were also measured, thus allowing us to calculate the Gibbs free energy and entropy of adsorption. Experimental results indicated that the adsorption of both proteins on butyl-containing adsorbents was exothermic, while their adsorption on octyl ones was endothermic. In addition, binding of both proteins with the butyl ligand is basically an adsorption process, while binding with the octyl ligand is adsorption and partition processes. Moreover, on both butyl or octyl, the adsorption enthalpy became increasingly positive as the ligand density increased, while the adsorption entropy became more positive as the alkyl chain length or density of the adsorbent increased. In addition, ITC was used to measure protein-protein interaction. The adsorption enthalpy of both proteins increased as the amount of bound protein increased, and the enthalpy increase of trypsinogen appeared to be higher than that of alpha-chymotrypsinogen A. This observation implies that protein-protein repulsion was stronger among trypsinogen molecules in the experiments.

Calorimetry↗

PubMed2000

Determination of catecholamines in pheochromocytoma cell (PC-12) culture medium by microdialysis-microbore liquid chromatography.

An in vitro microdialysis system was constructed for the measurement of catecholamines in pheochromocytoma cell culture medium. The novel microdialysis device is composed of a petri dish, a dialysis membrane and two transmission tubes. The dialysis membrane is located in the space of a petri dish such that it is immersed in the culture medium. Catecholamines contained in the culture medium diffused into a designed dialysis membrane with sufficient recovery (about 60%). Dialysates were collected by a sampling loop and introduced by an on-line injector to a microbore liquid chromatographic system for analysis of catecholamines. This assay yielded a detection limit of 0.2-0.5 pg/injection with acceptable intra- and inter-assay reproducibilities in 5 microl of dialysates. To evaluate the on-line microdialysis system, PC-12 cells were cultured in a petri dish within an incubator. The baseline concentration of dopamine in PC-12 cell culture medium was about 0.29 ng/ml which was elevated to 2.43 ng/ml after treatment with 0.5 mM potassium cyanide. In conclusion, the present microassay provides for the sensitive, direct measurement of catecholamines in culture medium while minimizing pretreatment procedures for sample preparation.

Animals↗

PubMed2000

ATP-stimulated c-fos and zif268 mRNA expression is inhibited by chemical hypoxia in a rat brain-derived type 2 astrocyte cell line, RBA-2.

The stimulus-transcriptional coupling during ischemia/hypoxia was examined for ATP-stimulated expression of immediate early genes (IEGs; c-fos, zif268, c-myc and nur77) in a rat brain-derived type 2 astrocyte cell line, RBA-2. Incubation of cells with 1 mM of extracellular ATP stimulated time-dependent expression of c-fos and zif268. ATP induced the largest increases in zif268 mRNA and a lesser one in c-fos mRNA. ATP also induced a slight increase in nur77 mRNA but was ineffective in inducing c-myc expression in these cells. Brief exposure of cells to potassium cyanide to simulate chemical hypoxia induced 9-fold and 7-fold transient increases in c-fos and zif268 expression, respectively, but did not affect c-myc or nur77 expression. When cyanide and ATP were added together, the expression of c-fos and zif268 expression was inhibited, and the effect was mimicked by simulating chemical hypoxia with sodium azide. To elucidate the mechanism involved, the effect of cyanide on ATP-stimulated increases in intracellular Ca(2+) concentrations, [Ca(2+)](i), and phospholipase D (PLD) activities were measured. Cyanide induced an increase in [Ca(2&plus);](i) and further enhanced the ATP-stimulated increases in [Ca(2+)](i) and PLD activities. Nevertheless, metabolic inhibitor, iodoacetate, blocked the ATP-induced c-fos and partially inhibited zif268 expression, and deprivation of cells with glucose also inhibited the ATP-induced c-fos expression. Taken together, these results demonstrate that both extracellular ATP and chemical hypoxia induce c-fos and zif268 expression in RBA-2 type 2 astrocytes. The chemical hypoxia inhibited ATP-stimulated c-fos and zif268 expression is not due to alterations in Ca(2+) and PLD signaling, and is at least partially related to metabolic disturbance in these cells.

Adenosine Triphosphate↗

PubMed2000

Finite element analysis of brain contusion: an indirect impact study.

The mechanism of brain contusion has been investigated using a series of three-dimensional (3D) finite element analyses. A head injury model was used to simulate forward and backward rotation around the upper cervical vertebra. Intracranial pressure and shear stress responses were calculated and compared. The results obtained with this model support the predictions of cavitation theory that a pressure gradient develops in the brain during indirect impact. Contrecoup pressure-time histories in the parasagittal plane demonstrated that an indirect impact induced a smaller intracranial pressure (-53.7 kPa for backward rotation, and -65.5 kPa for forward rotation) than that caused by a direct impact. In addition, negative pressures induced by indirect impact to the head were not high enough to form cavitation bubbles, which can damage the brain tissue. Simulations predicted that a decrease in skull deformation had a large effect in reducing the intracranial pressure. However, the areas of high shear stress concentration were consistent with those of clinical observations. The findings of this study suggest that shear strain theory appears to better account for the clinical findings in head injury when the head is subjected to an indirect impact.

Brain Injuries↗

PubMed2000

Blockage of amyloid beta peptide-induced cytosolic free calcium by fullerenol-1, carboxylate C60 in PC12 cells.

Amyloid beta protein (Abeta) alters signal transduction systems, including increases in the cytosolic free calcium ([Ca2+]i) response which have pathophysiological significance in Alzheimer's disease (AD). The purposes of this study were to elucidate the mechanism involved in Abeta's effect on the Ca2+ signal and to evaluate the effect of fullerenol-1, a water-soluble hydroxyl and superoxide radical scavenger, on the Abeta-induced Ca2+ response. Both Abeta and bradykinin (BK) dose-dependently elevated [Ca2+]i in PC12 cells. Fullerenol-1, at a concentration range between 100 nM and 1 microM, dose-dependently reduced the Abeta-induced [Ca2+]i response, but did not alter the subsequent BK-mediated process. Thapsigargin, an inhibitor of Ca2+-ATPase, released Ca2+ from the internal store and diminished the BK-mediated calcium spike but did not affect the Abeta-induced Ca2+ response. In the absence of extracellular calcium, the Abeta-induced, but not BK-induced, calcium spike was completely abolished. The Ca induced by Abeta did not enter through the voltage-dependent calcium channels or ligand gated calcium channels, because the peak of Abeta-evoked Ca2+ was not significantly altered by various Ca2+ channel blockers or a NMDA receptor antagonist MK801. In addition, neither cholera toxin nor pertussis toxin altered the Abeta-induced Ca response. The results demonstrated that Abeta-stimulated [Ca2+]i increase is due to Ca influx from an extracellular source rather than from the intracellular store. Alteration of the membrane lipid structure and permeability by free radicals generated by Abeta may be a major cause of Ca -influx. Furthermore, fullerenol-1, a novel antioxidant, may provide therapeutic benefits in neurodegenerative diseases such as AD.

Amyloid beta-Peptides↗

PubMed2000

Laser-induced acoustic emissions in experimental dental composites.

A laser thermoacoustic technique was innovated to evaluate laser-induced acoustic emissions (AEs) in experimental dental composites aged with 75% ethanol solution. Experimental composite systems of 75/25 BisGMA/TEGDMA resin filled with 0, 12.6, 30.0, and 56.5 vol% of 8-microm silanized and unsilanized BaSiO6 were analyzed. The sample size was 4.65 mm (diameter) x 0.5 mm (thick). Aging effects of immersing in 75% ethanol for up to 14 h on AEs were then evaluated. A continuous-wave CO2 laser was used to heat the samples. Acoustic emissions were collected as a function of filler fraction, laser power, silanization, and immersion time. Onset of burst-pattern acoustic signals characteristic of fracturing occurred at different laser powers for different tested groups. Acoustic emissions generally increased with laser power, in which lower laser powers produced low-amplitude (45-50 dB) signals; the amplitude distribution (50-85 dB) became more extensive as laser powers increased. After immersion, the lower laser powers could produce the same phenomenon. The higher the filler fraction, the fewer AEs generated. A large percentage AE reduction due to silanization was noted as a function of filler fraction. Unsilanized specimens showed more thermal damages than did silanized ones.

Acoustics↗

PubMed2000

Finite element analysis of thermo-debonding mechanism in dental composites.

Finite element method (FEM) has been extensively used for evaluating interfacial status inside biomaterials. This study using FEM was designed to evaluate the thermal stress behavior of a filler-matrix interface. The results were then compared to those of a previous study obtained by a laser thermoacoustic technique (LTAT). The experimental systems (75/25 Bis-GMA/TEGDMA resin reinforced with 0, 25, 50, and 75 wt% 8-microm silanized/unsilanized BaSiO6) as used in the previous study were modeled in this study. The established finite element models were based on coefficient of thermal expansion (CTE) Mismatch Phenomenon. The mechanical properties of the silane coupling agent, such as elastic modulus and thermal expansion coefficient used in the silanized model, were assumed to have optimal heat flux transfer. A third (imaginary) material was proposed to block the transfer of thermal stress between the filler and matrix in the unsilanized model. The thermal load simulation was based on steady-state thermal analysis. The results showed that: (1) The strain energy and interfacial shearing stress calculated from FEM validate the results from the previous LTAT study. (2) Comparing the stress distribution of silanized and unsilanized FEM models, the acoustic signals in LTAT study are mainly derived from debonding of the filler-matrix interface of silanized specimens, and from the matrix area of unsilanized specimens. Based on results to date, we conclude that the finite element method may be a powerful tool for exploring thermoacoustic mechanisms of dental composites.

Acoustics↗

PubMed2000

Amyloid beta peptide impaired carbachol but not glutamate-mediated phosphoinositide pathways in cultured rat cortical neurons.

Signal transduction systems, including cholinergic pathways, which are likely to be of pathophysiological significance are altered in Alzheimer's disease (AD). Muscarinic cholinergic receptors are linked to the hydrolysis of phosphoinositide, involving the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and the mobilization of cytosolic free calcium concentrations ([Ca2+]i). Effects of amyloid peptide (A(beta)) on these signals prior to neuronal degeneration were examined in cultured rat cortical cells. A(beta) increased the release of lactate dehydrogenase (LDH) in a concentration-dependent manner, however, it was blocked by B27 supplement. Prolonged exposure to a sublethal dose of A(beta) 25-35 or 1-42 disrupted carbachol-mediated release of Ins(1,4,5)P3 and [Ca2+]i, which was inhibited in media supplemented with B27 or the antioxidant vitamin E. In order to determine the specificity of the effect of A(beta), various agonists glutamate or KCl but not bradykinin which utilize the phosphoinositide cascade were investigated. Our results indicated that A(beta) did not affect the stimulation of glutamate or KCl-mediated production of Ins(1,4,5)P3 or cause elevation in [Ca2+]i. Furthermore, metabotropic agonist trans-1-amino-cyclopentane-1,3,-dicarboxylate (ACPD) elevated calcium level was not inhibited by A(beta) pre-treatment. Taken together, the results demonstrate that a sublethal dose of A(beta) selectively impaired cholinergic receptor-mediated signal transduction pathways, and antioxidant or B27 supplement attenuated this effect of A(beta). Alterations of cholinergic signaling by prolonged exposure to A(beta) could be involved in cortical neurodegeneration that occurs in AD. Because functional loss of cholinergic pathways is an important aspect of AD, the differences in susceptibility of these two types of receptors prior to other signs of A(beta) action is important and requires further investigation.

Amyloid beta-Peptides↗

PubMed2000

Fluoride ion diffusion from a glass-ionomer cement.

The aims of this study were to observe the release of fluoride ions (F-) from GC-Fuji Lining-LC(R) glass-ionomer cement, to assess the diffusion process, and to measure fluoride diffusivity (D) in the set cement. Specimens of various dimensions and shapes, ranging from discs to cylindrical rods, were fabricated for both open and embedded modes of testing. In the open mode studies, specimens with different surface to volume ratios were selected and immersed in 37 degrees C distilled water. In the embedded mode studies, only one diametral surface of the rod-shaped specimens of different lengths and diameters was exposed. F- concentration was measured using a fluoride electrode. The storage solution was analysed at predetermined intervals hourly, daily, and weekly (up to 10 weeks). Immediately after each sampling, the old storage solution was discarded and replaced with new distilled water. F- release from the set cements was detectable (0.4-3.8 ppm, varied with sample geometry), even after a 10-week sampling interval. F- release was greater in ground set cements (0.37 ppm/mg powder) than in control samples of unmixed powder (0.01 ppm/mg powder) immersed for 1 h. Two mechanisms for F- release were proposed. One was short-term and involved rapid dissolution from the cement surface. The other was more gradual and resulted in the sustained diffusion of ions through the bulk cement, which can be modelled by applying a mathematical technique known as separation of variables to Fick's Second Law of Diffusion. The mean D of F- in embedded set cements of glass-ionomer was (1.4+/-0.5)x10-11 cm2/s, with higher apparent D observed in open mode samples [(7.6+/-1.4)x10-11 cm2/s].

Diffusion↗

PubMed2000

Influence of silanization and filler fraction on aged dental composites.

The effect of silanization and filler fraction on the mechanical properties of aged dental composites was investigated. Experimental composites (75/25 Bis-GMA/TEGDMA resin reinforced with 0, 12.6, 30.0, and 56.5 vol% 8 microm silanized/unsilanized BaSiO6) were fabricated into 4.7 mm diameter x 2.2 mm thick discs and 3.5 mm diameter x 7.3 mm thick discs for diametral tensile and compressive tests, respectively. The effect of immersion in 75% ethanol at 37 degrees C for 0-30 days on the diametral tensile strength (DTS) and compressive strength (CS) of the samples was evaluated and analysed by ANOVA and Tukey LSD test. The fracture interface between filler and resin matrix was then examined by scanning electron microscope. Results and subsequent statistical evidence from DTS (18.6+/-7.6 MPa, silanized versus 11.7+/-2.6 MPa, unsilanized) and CS (85.1+/-29.7 MPa, silanized versus 56.0+/-11.3 MPa, unsilanized) strongly implies that silanization may greatly enhance the mechanical properties of the resin composites. Furthermore, it also shows that both DTS and CS increased proportionally as the filler fraction of the composites increased. However, in the unsilanized groups, DTS decreased (up to 40%) as the filler fraction increased, and CS showed no relevance to the filler fraction at all. As for the influence of aging, it was found that both DTS and CS showed a significant decrease after immersion in 75% ethanol, and silanization heavily correlated with the filler fraction of aged-resin composites. Microscopic examination of the fractured samples showed that failure primarily occurred within the resin matrix per se for silanized composites and adjacent to the filler particles for unsilanized composites. All the evidence points to the conclusion that mechanical properties of aged-resin composites can be greatly influenced by silanization and the filler fraction.

Analysis of Variance↗

PubMed2000

Acoustic emissions generated in aged dental composites using a laser thermoacoustic technique.

The heating up of dental composites by laser will produce acoustic emissions (AEs) that may be related to fracture mechanisms in the composites. It has been proved that the mechanical properties of dental composites are affected by storage in food simulating liquids, i.e. 75% ethanol, which has a solubility parameter approximating to that of bisphenol glycidyl dimethacrylate (BisGMA) resin. A new method was innovated to evaluate the laser-induced AEs in dental composites aged by 75% ethanol solution. Model systems (50/50 BisGMA/TEGDMA resin filled with 0% and 75 wt.% 5-10 microm silanized BaSiO6) as well as three commercial composites (Marathon One, Z100 and Herculite XRV) were used in this study. Nine samples acting as the control group were tested to establish the correlation of AEs to laser power. The effect of ageing by immersion in 75% ethanol on AEs and diametral tensile strength (DTS) was then evaluated. A quasi-continuous wave CO2 laser was used to heat up the composites. AEs of frequency 100-200 kHz were collected, filtered, recorded and processed using a 4610 Smart Acoustic Monitor. Burst patterns, which formally were assumed to be correlated to fracture mechanisms, were also identified from the data obtained at laser power > or = 5 W for commercial composites and > or = 4 W for model systems. Higher laser powers cause the AE to increase for all composites except unfilled model resin. AEs as a function of power for all aged systems were flat (< 100 events) below 4 W. Emissions then rose sharply to > 1000 events at 7.1 W. Statistically significant differences were found between the AEs obtained at 5 W (commercial composites) and those at 4.3 W (model systems) for material systems and storage times. Marathon One was less affected by the laser and an abrupt change in AE was found between days 0 and 7 of storage for all commercial composites. The AE value from the unfilled model resin was found to be significantly different from that of the model composites. However, they showed an increase in AEs with length of storage time, which was inversely associated with the decreased tendency of their immersed DTS values. Laser-induced AEs may be a valuable adjunct to conventional mechanical testing.

Acoustics↗

PubMed2000

Effect of temperature and flux concentration on soldering of base metal.

The present study used the acoustic emission (AE) technique to evaluate interactions among soldering temperature, flux treatment, and the resultant ultimate tensile strength (UTS). Scanning electron microscopy (SEM) was used to examine fracture surfaces of the solder joints. Specimens were cast from removable partial denture alloy and then placed in a jig with a gap distance of 1.0 mm. A high-frequency soldering machine with an optical pyrometer was used for soldering at 1150 degrees C and 1200 degrees C, respectively. The flux concentrations were 67% and 75%. The soldered specimens were subjected to tensile test at a crosshead speed of 0.05 mm/min. During testing, acoustic emissions in the frequency range of 100--1200 kHz were collected, filtered, recorded, and processed by a sensing device. The results were analysed by ANOVA and Tukey LSD test. UTS at different temperatures showed no significant difference according to either mechanical or acoustic results. But in the 1200 degrees C group, the UTSs and AE counts showed significant differences (P<0.05) at both flux concentrations. SEM showed that the 1200C group had better dendritic crystal structure than did the 1150 degrees C group. In the 1200 degrees C group specimens with 67% flux had fewer flux inclusion bodies and dendritic crystals than did specimens with 75% flux. The 75% flux subgroup produced high-amplitude (60--70 dB) acoustic signals within the elastic deformation zone, while the 67% flux subgroup produced similar signals within the plastic deformation zone, either beyond the 0.2% yield point or before fracture.

Acoustics↗

PubMed2000

Assessing the implant/bone interface by using natural frequency analysis.

OBJECTIVE: A number of techniques have been proposed for detecting the stability of dental implants. However, the clinical applicability of those methods is still limited. The purpose of this study was to evaluate a new innovative, noninvasive, minimum-contact method for the stability assessment of dental implants. STUDY DESIGN: Natural frequency is a physical property of a structure, which is strongly related to its boundary conditions. In this study, a modal testing technique was carried out to measure the natural frequency of dental implants. The implants were fixed by a metal clamp stand and were excited to vibrate by an impulse hammer. A noncontact piezoelectric microphone then acoustically acquired the vibration responses of the implants. Natural frequencies of the tested implants were recorded under various clamping forces and clamping levels. RESULTS: Natural frequencies of the tested implants were concentrated from 8 to 19 kHz under different boundary conditions. On the other hand, the natural frequency values decreased when boundary levels and boundary force were reduced. Linear relationships (P <.005) were found between response frequencies and the degree of implant stability. CONCLUSIONS: Our results show that the boundary status of an implant can be monitored by detecting its natural frequency. A noncontact transducer used in this study can also serve as a useful tool for future clinical investigations.

Dental Implants↗

PubMed2000

In vivo and in vitro natural frequency analysis of periodontal conditions: an innovative method.

BACKGROUND: When measuring periodontal disease, various types of equipment for making objective measurements of tooth mobility have been proposed. However, these devices and methodology are insufficient in terms of reliability. An innovative method using vibrational theories to assess the periodontal attachment level of natural teeth is presented in this study. METHODS: Modal testing technique, a non-destructive and time-saving method, was used for non-invasive and quantitative measurement of the natural frequencies of the upper central incisor in vivo and in vitro. A finite element model was established, and modal testing experiments were simulated to assess the relationship between bone level and teeth. RESULTS: The first dominant natural frequency of healthy human upper central incisors ranges from 710 Hz to 3,360 Hz, with an average of 1,701+/-679 Hz. Both in vitro experiment and finite element simulation showed that lowering of the attachment level causes a significant decrease in the natural frequencies, and a strong relationship (r = -0.99, P <0.01) was observed between the frequency and the bone level height. CONCLUSIONS: Our results suggested that natural frequency is an important parameter for assessing the periodontal condition and that the modal testing technique is a useful supplemental method for assessing the attachment conditions of the periodontium.

Acceleration↗

PubMed2000

[Construction, expression and activity test of a reshaping single-chain antibody against human CD3].

Monoclonal antibody (McAb) against human CD3 can adjust human body's immune statement in various ways, so that its clinical potential is highly regarded. In order to overcome the immunogenecity related to the murine McAb, this research effort was focused on constructing a reshaping single-chain antibody(scFv) against human CD3 employing antibody engineering. First, the CDRs of the murine McAb against human CD3 OKT3 was transplanted into the light-chain framework regions (FRs) of human McAb LS1 and the heavy-chain FRs of human McAb Nd respectively, spatial conformation was predicted by computer analysis. Then some particular residues were replaced in FRs basing on the result of conformational prediction to draw out the amino acid sequences of the reshaped VL and VH. The genes were chemically synthesize and inserted into an expression vector pROH80 to construct the reshaping scFv. Inducing the expression of reshaping scFv, the products are mainly as inclusion bodies. The reshaping scFv was expressed in another vector pALM. The inclusion bodies were denatured and then renatured by gel filtration. The renatured products were purified by immobilized metal affinity chromatograph (IMAC). Finally, the antigen-binding activily of the reshaping scFv against human CD3 was testified by the Compelitire in hibilory fluorescenceactivated cell sorting (FACS). The competitive inhibition rate is 18%.

Amino Acid Sequence↗

PubMed1999

Selective subcellular redistributions of protein kinase C isoforms by chemical hypoxia.

The mechanisms of neuronal degeneration following hypoxia/ischemia remain undefined, but the processes include increases in neurotransmitter release, elevation of cytosolic-free calcium concentration, and changes in signal transduction pathways. Activation of the multigene family of protein kinase C (PKC) has been associated with the release of neurotransmitter and the survival of neurons. Therefore, to understand which PKC isozymes are involved in hypoxia/ischemia-induced neuronal degeneration, we examined PKC isozymes after chemical hypoxia (i.e., KCN exposure) in PC12 cells. Cell toxicity, as measured by lactate dehydrogenase (LDH) release, was increased significantly by KCN in glucose-free DMEM and was exaggerated by acute 12-O-tetradecanoyl phorbol-13-acetate (TPA) pretreatment. Under parallel conditions, KCN elevated cytosolic-free calcium ([Ca2+]i) in glucose-free but not in glucose containing DMEM, and TPA pretreatment did not exaggerate KCN's effect on [Ca2+]i. Thus, increases in [Ca2+]i are not sufficient for the synergistic toxic effect of KCN and TPA. In the glucose-free DMEM, selective PKC isozyme inhibitor Go 6976 at 10 nM completely inhibited KCN-induced LDH release and at higher concentrations (1 microM) inhibited the basal levels of LDH release. The protein levels of PKCs in the nuclear, membrane, and cytosolic fractions were measured by Western blot analysis using antibodies against specific isoforms. Two Ca2+-dependent (-alpha, -gamma) and four Ca2+-independent (-delta, -epsilon, -zeta, and -lambda) isozymes were identified and two isozymes (-beta and -theta) were not detected in the subcellular fractions of PC12 cells. Treatment of the cells with TPA significantly activated translocation of conventional PKC-gamma from the cytosol to the membrane and nuclear fractions and other PKC isozymes (-alpha, -delta, and -epsilon) from the cytosol to the membrane, but not atypical PKC-zeta and -lambda. Although only the levels in the nuclear PKC-gamma but not other PKC isozymes were increased significantly following KCN, the levels of cPKC-alpha and -gamma in the membrane mainly- and those and PKC-epsilon in the nucleus-were increased when KCN was combined with TPA. In addition, this condition (TPA + KCN) did not affect the TPA insensitive atypical isozymes, PKC-zeta or -lambda. Taking the results together, differential activation/translocation of PKC isozymes by KCN and TPA is important in the regulation of chemical hypoxia-induced cell injury in PC12 cells.

Animals↗