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Biomedical subjects

R C Chang

Publications and source records attributed to R C Chang.

At least 37 records · Page 2Linked to original sources
PubMed1999

Technical note: a new model for quantitative analysis of brain oedema resolution into the ventricles and the subarachnoid space.

OBJECTIVE: The aim of the current study was to develop an experimental animal model for quantitative analysis of oedema resolution via the subarachnoid space and the ventricular system using fluorescent oedema markers. METHODS: Artificial cerebrospinal fluid (CSF) containing TRITC-albumin (MW 67.000D) and Na(+)-fluorescein (MW 376D) was continuously infused into the white matter of the left frontal lobe of New Zealand white rabbits (n = 6) at a rate of 100 microliters/h for 3 hrs. A closed cranial window for superfusion of the brain surface with artificial CSF fluid (3 ml/h) was implanted above the left parietal cortex for measurement of the fluorescence markers in the subarachnoid space. Uptake of the fluorescence indicators into the ventricles was quantified by ventriculo-cisternal perfusion (3 ml/h). The effluates were collected at 30 min intervals for 3 hrs after the start of infusion. Clearance of the oedema fluid into the perfusates was measured by fluorescence spectrophotometry. RESULTS: At an intracranial pressure of 15.0 +/- 1.7 mm Hg (mean +/- SEM) both indicators started to accumulate in the subarachnoid and ventricular perfusates at 90 min following onset of oedema fluid infusion. The concentrations of the indicators in the ventricular system increased to 7.7 +/- 5.1% of Na(+)-fluorescein and 16.1 +/- 13.0% of TRITC-albumin of the total amount infused were recovered in the ventricular system at 3 hours after start of the oedema infusion, while 3.4 +/- 3.2% of Na(+)-fluorescein and 3.7% +/- 3.2 of TRITC-albumin, respectively, were found in the effluates of the subarachnoid space. CONCLUSION: The present study demonstrates that resolution of vasogenic brain oedema into the cerebral ventricular system and the subarachnoid space following its entry into cerebral white matter can be quantitatively analysed using fluorescence markers, which serve as oedema fluid indicators. The results indicate that the oedema fluid is cleared not only into the ventricular system but also via the subarachnoid space.

Animals↗
PubMed1999

Using three-dimensional-computerized tomography as a diagnostic tool for temporo-mandibular joint ankylosis: a case report.

Roentgenographic examination has long been a useful diagnostic tool for temporo-mandibular joint (TMJ) disease. The methods include TMJ tomography, panoramic radiography and computerized tomography (CT) scan with or without injection of contrast media. Recently, three-dimensional CT (3D-CT), reconstructed from the two-dimensional image of a CT scan to simulate the soft tissue or bony structure of the real target, was proposed. In this report, a case of TMJ ankylosis due to traumatic injury is presented. 3D-CT was employed as one of the presurgical roentgenographic diagnostic tools. The conventional radiographic examination including panoramic radiography and tomography showed lesions in both sides of the mandible. CT scanning further suggested that the right-sided lesion was more severe than that on the left. With 3D-CT image reconstruction the size and extent of the lesions were clearly observable. The decision was made to proceed with an initial surgical approach on the right side. With condylectomy and condylar replacement using an autogenous costochondral graft on the right side, the range of mouth opening improved significantly. In this case report, 3D-CT demonstrates its advantages as a tool for the correct and precise diagnosis of TMJ ankylosis.

Ankylosis↗
PubMed1999

Coronal approach for the replacement of the condylar head in bilateral temporomandibular joint ankylosis: report of three cases.

Limitation of mouth opening has long been the chief complaint for patients who suffer from temporomandibular joint (TMJ) ankylosis. For surgical treatment of this disease, several ways have been employed for access to the condylar fossa, including the preauricular, postauricular, perimeatal, endaural and Risdon approaches. In this article, we report three patients with bilateral TMJ ankylosis who underwent replacement of both condylar heads using the bicoronal approach. The advantages demonstrated with this surgical technique are ease of access to the condylar head, ideal surgical exposure field, utility of temporalis muscle and fascia and minimal risk of facial paralysis.

Adult↗
PubMed1998

Relevance of calcium homeostasis in glial cell swelling from acidosis.

Tissue acidosis from trauma or ischemia induces cytotoxic brain edema, mainly affecting astrocytes. In vitro, lactacidosis induces a dose-dependent swelling of glial cells. Activation of membrane transporters and channels, also involved in regulation of intracellular pH (pHi), has been identified as underlying mechanism, although details are poorly understood. We have currently studied whether Ca(2+)-ions play a role in acidosis-induced glial swelling and the associated intracellular acidification. The medium pH of a cell suspension (C6 glioma) was lowered from control (7.4) to 6.2 by lactic acid. Cell volume (CV) and pHi were assessed by flow cytometry. During acidosis in normal medium (2.2 mM Ca2+) CV reached a maximum of 125.1%. In a calcium-free medium swelling from acidosis was inhibited by 74%, while additional buffering of intracellular calcium (Ca2+i) by BAPTA-AM had no further effect. Buffering of Ca2+i alone did not affect the CV increase from acidosis at all. pHi which is decreasing during acidosis was not influenced by the above modifications. The present experiments indicate that lactacidosis-induced glial swelling depends on the presence of extracellular Ca(2+)-ions, while alterations of Ca2+i do not seem to be involved.

Acid-Base Equilibrium↗
PubMed1997

Swelling and damage of glial cells by lactacidosis and glutamate: effect of alpha-trinositol.

The therapeutical efficacy of alpha-trinositol (D-myo-inositol-1,2,6-trisphosphate), an isomer of the intracellular messenger IP3, was analyzed on cytotoxic swelling and damage of glial cells in vitro from lactacidosis or glutamate. C6 glioma cells suspended in a physiological medium were either exposed to pH 5.0 by administration of lactic acid, or to 1 mM glutamate. Cell swelling and viability were quantified by flow cytometry. Lactacidosis of pH 5.0 led to an increase in cell volume to 139.7 +/- 1.3% within 20 min whereas alpha-trinositol was reducing the swelling response by approximately 25% (P < 0.01). In addition, at pH 5.0 the fraction of viable cells was lowered from 94.3 +/- 0.2% (control) to only 53.8 +/- 3.1% after 60 min. Alpha-trinositol was found to protect also cell viability; at 60 min of lactacidosis 70.2 +/- 1.6% of the cells still were viable (P < 0.01). The addition of glutamate (1 mM) to the cell suspension led to a steady increase in cell size, reaching 110% of control at 120 min, irrespectively of whether alpha-trinositol was added or not.

Acidosis, Lactic↗
PubMed1997

Role of calcium ions in acidosis-induced glial swelling.

Tissue acidosis occurring in cerebral ischemia and traumatic brain injury is a mediator of cytotoxic brain edema. In vitro, extracellular lactacidosis induces swelling of glial cells in a dose dependent manner. pH-regulatory membrane transporters and channels have been identified which are involved in the increase of the glial cell volume. Underlying mechanisms of their activation are poorly understood, however. We have, therefore, addressed the question, whether and how Ca(2+)-ions play a role in acidosis-induced glial swelling and intracellular acidification. For that purpose C6 glioma cells were suspended and the pH in the medium was lowered from 7.4 (baseline) to 6.2 by isotonic lactic acid. Cell volume and intracellular pH (pHi) were assessed by flow cytometry. In the presence of Ca(2+)-ions the cell volume reached a maximum of 125.1% from acidosis. In experiments using a calcium-free suspension medium, cell swelling from acidosis was inhibited by 74%. Additional buffering of intracellular calcium (Ca2+i) had no further inhibitory effect on acidosis-induced cell swelling, while buffering of Ca2+i by BAPTA-AM alone did not affect the glial volume increase secondary to administration of lactic acid. pHi which was decreasing from acidosis was not affected by the experimental modifications of the Ca(2+)-concentration in the medium or cytosol. The present data indicate that lactacidosis-induced glial swelling depends on the presence of extracellular Ca(2+)-ions, while release of Ca(2+)-ions from intracellular stores does not seem to be involved.

Acidosis↗
PubMed1997

Effect of alpha-trinositol on swelling and damage of glial cells by lactacidosis and glutamate.

The therapeutic efficacy of alpha-trinositol (D-myo-inositol-1,2,6-trisphosphate), an isomer of the intracellular messenger IP3, was analyzed for cytotoxic swelling and damage of glial cells in vitro from lactacidosis or glutamate. Lactacidosis and the interstitial accumulation of glutamate are prominent sequelae in ischemic or traumatic brain tissue. C6 glioma cells harvested from culture and suspended in a physiological medium were either exposed to pH 5.0 by administration of lactic acid, or to 1 mM glutamate at normal pH. Cell swelling and viability were quantified by blood flow cytometry. Addition of alpha-trinositol (3 mM) under control conditions at pH 7.4 resulted in transient cell shrinking to 96.5 +/- 1.3% of control within 3 min (p < 0.05). Lactacidosis of pH 5.0 led to an increase in cell volume to 139.7 +/- 1.3% within 20 min, whereas alpha-trinositol reduced the swelling response by approximately 25% (p < 0.01). In addition, cell viability was severely affected at pH 5.0 amounting to only 53.8 +/- 3.1% after 60 min. alpha-Trinositol was found to markedly improve cell viability; at 60 min 70.2 +/- 1.6% of the cells were still viable (p < 0.01). Addition of glutamate (1 mM) led to a steady increase in cell size, reaching 110% of control after 120 min, irrespective of wether alpha-trinositol was present or not. The attenuation of cell swelling may be attributed to an interference with pH-regulatory mechanisms, such as the Na+/H(+)-antiporter, while protection of cell viability might be caused be effects of alpha-trinositol on Ca(2+)-overload. On the other hand, the increase in cell volume by glutamate associated with its intracellular uptake was not influenced by alpha-trinositol.

Acidosis, Lactic↗
PubMed1997

Role of protein kinase C in acidosis induced glial swelling--current understanding.

A major factor in secondary brain injury following cerebral trauma is accumulation of lactic acid resulting in glial swelling. Further, evidence obtained in this context demonstrates activation of protein kinase C (PKC) under these circumstances. Glial swelling from acidosis is attributable to activation of the Na+/H(+)-exchanger, mediating influx of Na(+)-ions in exchange for the extrusion of H+ ions. The antiporter is activated following phosphorylation by PKC. The current study was made to elucidate the role of PKC activation in acidosis-induced glial swelling. For that purpose, suspended C6 glioma cells were used to examine changes of the cell volume and intracellular pH (pHi). Acidosis was induced by administration of isotonic lactic acid. Stimulation of PKC by the phorbol-ester PMA was significantly enhancing glial swelling from severe acidosis (pH 6.2), whereas the decrease of pHi was somewhat attenuated. On the other side, inhibition of PKC by staurosporine did not affect cell swelling nor the decrease of pHi from acidosis. The results indicate that activation of PKC in cerebral trauma or ischemia may enhance glial swelling from lactacidosis.

Acidosis, Lactic↗
PubMed1997

Effect of mild and moderate hypothermia on the acidosis-induced swelling of glial cells.

The effect of mild (32 degrees C) and moderate (27 degrees C) hypothermia was analyzed on the cell volume and intracellular pH (pHi) of C6 glioma cells at normal pH and during lactacidosis at pH 6.2 in vitro. The cells were suspended in an incubation chamber under continuous control of pH, PO2 and temperature. Cell swelling was quantified by an advanced Coulter-system. pHi was measured by flow cytometry using the fluorescent dye bis-carboxyethyl carboxyfluorescein (BCECF). Following a control period at 37 degrees C, the ambient temperature was decreased to 32 degrees C for 30 min, and subsequently to 27 degrees C for another 30 min. Hypothermia alone led to an immediate and significant cell volume increase of 107.3 +/- 0.4% (mean +/- SEM) of control after 30 min at 32 degrees C, and further swelling to 110.5 +/- 0.9% after 30 min at 27 degrees C. Yet, hypothermia (27 degrees C) afforded partial protection against the acidosis-induced cell swelling at pH 6.2, which was reaching to 120.4 +/- 0.9% in the normothermic control group after 60 min, while only to 111.3 +/- 0.9% at 27 degrees C. Hypothermia, however, was associated with a more pronounced decrease of the pHi during acidosis (6.3 +/- 0.04) as compared to that of the normothermic control falling then to 6.5 +/- 0.03. The results demonstrate that mild and moderate hypothermia induce glial cell swelling, but simultaneously inhibit cell swelling from acidosis. The protection against cell swelling, however, has its price as indicated by the enhancement of the intracellular acidification.

Acidosis, Lactic↗
PubMed1997

Quantitative analysis of brain edema resolution into the cerebral ventricles and subarachnoid space.

Resolution of vasogenic brain edema was examined using a model of infusion of fluid into the brain of rabbits. For this purpose infusion of Texas Red-albumin (MW 67.000 D) and sodium fluorescein (MW 376 D) dissolved in artificial cerebrospinal fluid (mock CSF) was made into the white matter of the left frontal lobe of the brain. In order to quantify the portion of edema fluid which was cleared by the ventricular system, a ventriculo-cisternal perfusion was performed with mock CSF. A closed cranial window was implanted above the left parietal brain for superfusion of the cerebral cortex with mock CSF, in order to study resolution of the artificial edema fluid via the subarachnoid space. CSF-samples were collected in 30 minutes-intervals and analysed with a spectrophotometer. The clearance of edema fluid was examined under low (2-5 mmHg) and medium (9-12 mmHg) intracranial pressure (ICP). In the low-pressure group both edema fluid markers were found in the ventriculo-cisternal and subarachnoid perfusate at 60 min and 90 min, respectively, after start of infusion. In the group with moderately increased ICP the markers appeared at 90 min and 120 min, respectively. The amount of clearance of fluorescent dye via the subarachnoid space was the same in both groups and independent of the intracranial pressure.

Animals↗
PubMed1996

Studying the active site pocket of Staphylococcus hyicus lipase by site-directed mutagenesis.

Site-directed mutagenesis of a previously constructed, recombinant Staphylococcus hyicus lipase (49 kDa) showed that Val363 played a role in catalysis and substrate-binding. In comparison with wild type enzyme, the 64% and 89% decrease in the catalytic efficiency (kcat/Km) of the V363N and V363A enzymes, respectively, were largely caused by a 3.5- and 5.5-fold increase in the substrate-binding affinity (Km), respectively. In comparison with wild type enzyme, a G371A enzyme showed a 40% decrease in the Km, suggesting that G371 was important for substrate-binding specificity. Site-directed mutagenesis of the active site Asp559 revealed that in comparison with wild type enzyme, a D559E enzyme exhibited a 47% decrease in the kcat/Km but a twofold increase in the Km for p-nitrophenyl butyrate, suggesting that Asp-559, a component of the catalytic triad, was involved in substrate-specificity.

Binding Sites↗
PubMed1996

Facile purification of highly active recombinant Staphylococcus hyicus lipase fragment and characterization of a putative lid region.

A fragment of Staphylococcus hyicus lipase gene (Ala248 to Ala640) was inserted into plasmid pET20(b+). The resulting His-tagged recombinant protein (49 kDa) was overexpressed in Escherichia coli BL21(DE3) as an highly active lipase and was purified by nickel-coupled resin. Site-directed mutagenesis showed that in comparison with wild type enzyme, the L326F and L326A enzymes showed a 3.4 and 5 fold increase in the K(m), respectively, but only a 44% and a 64% decrease in the kcat/K(m), respectively, suggesting that Leu326 of the putative lid participated in substrate-binding.

Amino Acid Sequence↗
PubMed1996

Characterization of the ferrous ion binding sites of apple 1-aminocyclopropane-1-carboxylate oxidase by site-directed mutagenesis.

The putative ferrous ion binding sites (H177, D179, and H234) of apple fruit 1-aminocyclopropane-1-carboxylate oxidase determined by homology comparisons with enzymes which required non-haem Fe2+ for activity were altered by site-directed mutagenesis. The activities of mutants (H177F, D179H, D179A, H234F, and H234D) were completely lost. SDS-PAGE and western immunoanalysis confirmed that loss of enzyme activity in mutants was not due to impaired enzyme expression. These results strongly suggest that H177, D179, and H234 are the Fe2+ binding site.

Amino Acid Oxidoreductases↗
PubMed1996

Site-directed mutagenesis of a novel serine arylesterase from Vibrio mimicus identifies residues essential for catalysis.

Site-directed mutagenesis (SDM) of an arylesterase (the arylesterase) from Vibrio mimicus revealed that residues S29, H153, and D96 constituted a catalytic triad. The use of a serine residue for ester hydrolysis by the arylesterase proves that the enzyme is a novel serine arylesterase. SDM also showed that D28 was necessary for the esterase activity; to our knowledge it is the first time that a residue immediately preceding the active-site serine in esterases was shown biochemically to possess such a property. The results further suggest that D28 plays a role in substrate-binding. Residue 31 was firmly shown to participate in the binding of N-acetyl-D, L-phenylalanine beta-naphthyl ester (NAPNE), an artificial substrate for chymotrypsin. The S31G enzyme showed a 4 fold decrease in the Km for NAPNE over that of wild type enzyme, proving residue 31 is important for substrate-specificity. A mechanism for binding and catalysis of esters by the arylesterase is proposed, which includes the unique role of S31 for aromatic (hydrophobic) acyl-binding. The biochemical properties of the arylesterase suggest that the enzyme stands out as a member of a distinct subfamily within a recently proposed, lipolytic enzyme family.

Amino Acid Sequence↗
PubMed1996

Swelling, intracellular acidosis, and damage of glial cells.

Cerebral ischemia and severe head injury among others are associated with a limited availability of oxygen, leading to cell catabolism as well as anaerobic glycolysis. Resulting metabolites, such as arachidonic- and lactic acid, can be expected to leak into perifocal brain areas, contributing there to cytotoxic swelling and damage of neurons and glia. Since elucidation of mechanisms underlying cell swelling and damage in the brain is difficult in vivo, respective investigations were carried out in vitro using suspended glial cells. Thereby, effects of arachidonic acid (AA) and of lactacidosis on glial cell volume, intracellular pH (pHi), and cell damage were analyzed utilizing flow cytometry. AA led to an immediate, dose dependent swelling and intracellular acidosis of glial cells. A concentration of 0.1 mM increased cell volume to 110% of control and decreased pHi to 7.05. Whereas glial swelling was permanent, pHi recovered to baseline after 90 min. Cell viability of 90% remained unchanged after addition of AA up to 0.1 mM, while at 0.5 mM it was significantly decreasing. Glial swelling from AA was nearly completely inhibited by the aminosteroid U-74389F or by using a Na(+)-free suspension medium for the experiment. Acidification of the medium to pH 6.8 or 6.2 led to a cell volume of 110% or 120% of control without affecting cell viability. The cells were not capable to defend their normal pHi during lactacidosis of the suspension medium but became acidotic as well. Addition of amiloride or utilization of Na(+)-free medium inhibited cell swelling from lactacidosis, while intracellular acidosis was even more pronounced. The results indicate that AA as well as acidosis are potent mediators of glial swelling and damage at levels found under pathophysiological conditions in the brain in vivo. Whereas intracellular acidification caused by AA was reversible, glial cells were unable to regulate their pHi during maintenance of extracellular acidosis. Concerning the mechanisms of glial swelling by AA, the production of oxygen- and lipid radicals might play a major role in the swelling process. The results indicate a role of the Na+/H(+)-antiporter in acidosis-induced glial swelling, whereas the exchanger has a limited significance for maintenance of pHi. As seen, the final pathway of glial swelling from both, AA and lactacidosis, requires a net influx of Na(+)-ions, probably together with Cl-ions, and osmotically obliged water.

Acid-Base Equilibrium↗
PubMed1996

Premedication with diclofenac and prednisolone to prevent postoperative pain and swelling after third molar removal.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) and steroid are able to effectively reduce the postoperative sequelae after impacted third molar removal. However, few studies have evaluated the activity of the combined effects of these two drugs. The purpose of this study was to evaluate whether a single dose of prednisolone, taken orally before operation, would increase the effects of diclofenac in preventing pain and swelling after surgical removal of impacted third molars. METHODS: Thirty healthy patients requiring surgical removal of four third molars were randomly assigned to two groups. In group A, patients were preoperatively given 50 mg of either diclofenac or placebo when impacted teeth were removed on each side. In group B, in addition to 50 mg of diclofenac, 10 mg of prednisolone or placebo was administered. Postoperative pain, swelling and range of mouth opening were recorded. RESULTS: Preoperative administration of 50 mg of diclofenac could relieve pain and swelling more than the placebo. Additional 10 mg of prednisolone could further reduce swelling. CONCLUSIONS: The combination of diclofenac and prednisolone has a better analgesic and anti-inflammatory effect.

Adult↗
PubMed1995

Vibrio mimicus arylesterase has thioesterase and chymotrypsin-like activity.

A Vibrio mimicus serine arylesterase and an Escherichia coli thioesterase/serine protease share 49.4% amino acid identity. The arylesterase has thioesterase activity for benzoyl-CoA and chymotrypsin-like activity for N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (NBPNPE). The gene encoding the V. mimicus enzyme is designated etpA. Substituting Ser31 of the V. mimicus enzyme with a glycine or an alanine altered its activity. In comparison with wild type enzyme, the S31A enzyme showed a 5-fold increase and 57% decrease in the catalytic efficiency for benzoyl-CoA and NBPNPE, respectively, and the S31G enzyme showed a 3.6-fold increase and 43% decrease in the catalytic efficiency for benzoyl-CoA and NBPNPE, respectively. For the two mutant enzymes an 8-fold decrease and a 6- to 7-fold increase in Km were seen for benzoyl-CoA and NBPNPE, respectively. The mutagenesis results prove that residue 31 plays an important role in the substrate-specificity.

Acyl Coenzyme A↗