Search PubMed⌕ Search

Biomedical subjects

D T Chiu

Publications and source records attributed to D T Chiu.

At least 73 records · Page 4Linked to original sources
PubMed1993

Direct alteration of erythrocyte membrane properties by 1-chloro-2,4-dinitrobenzene without oxidant challenge.

The electrophilic agent, 1-chloro-2,4-dinitrobenzene (CDNB), has been widely used as an intracellular glutathione-depleting agent. However, its possible effect on the functional integrity of cell membrane has largely been neglected. Incubation of human erythrocytes (RBC) with various concentrations of CDNB (0.5 to 5.0 mM) in potassium-free, phosphate buffered saline containing ouabain resulted in a drastic depletion of cellular glutathione as well as a dose-dependent increase in passive potassium leakage. Further, an osmotic gradient ektacytometry profile indicated that the deformability index (DI) of CDNB-treated RBC was substantially lower than the DI value of the control. Also, CDNB caused a dose-dependent increase in the rate of shear-induced fragmentation of resealed ghost prepared from treated, intact erythrocytes. These CDNB-induced changes were accompanied by stomatocytic transformations as evidenced by scanning electron micrographs. Additional study indicated that CDNB caused a dose-dependent decrease in thiol concentrations of RBC membrane. SDS-PAGE analysis of membrane proteins revealed new Coomassie blue stainable bands, most noticeable below band-7 (M.W. 20,000). The effects of CDNB on RBC deformability and membrane proteins were also investigated under an atmosphere without oxygen (under nitrogen) and similar effects were observed between that under room air and that under nitrogen. Taken together, these data strongly indicate that CDNB has an adverse effect on the RBC membrane integrity in addition to its ability to deplete intracellular glutathione, possibly through its interaction with membrane sulfhydryl groups.

Dinitrochlorobenzene↗
PubMed1992

A new mutation responsible for severe G6PD deficiency in two ethnic Chinese with different clinical presentations: determination by a direct PCR sequencing technique.

To test the hypothesis that clinical manifestation in G6PD deficiency correlates with a molecular lesion, we investigated the G6PD gene of two Chinese Americans both of whom had G6PD deficiency, but who manifested different clinical presentations. In this study, we have developed a direct PCR sequencing protocol to examine the human G6PD gene. By using optimized PCR conditions with internal primers, we were able to amplify a 4.2 kb DNA fragment (covering exon 3 through 13 of the G6PD gene) of consistently high quality. From this we were then able to generate high quality single-stranded DNA templates by asymmetric PCR for subsequent sequencing. We also overcame the crossband problem by using internal primers, high temperature reaction with Taq I DNA polymerase, and/or sequencing with gene 32 protein. We could consistently amplify exons 1 and 2 despite their high G/C content by substituting 75% of dGTP with deoxy-7-deaza-guanosine triphosphate. By using this novel approach, we have identified a new mutation at cDNA position 1376 from G to T, which causes substitution of Leu for Arg at amino acid position 459. This mutation has not been reported in other ethnic groups. It is the only genetic defect in the coding regions of the G6PD gene of these two G6PD deficient individuals. We speculate that in addition to a defect in the G6PD gene, other factors also play a role in the clinical manifestation of G6PD deficiency.

Base Sequence↗
PubMed1991

Two commonly occurring nucleotide base substitutions in Chinese G6PD variants.

Using a direct PCR sequencing technique, we have identified two DNA base substitutions in 8 different biochemical G6PD variants of Chinese origin. Neither one of these abnormalities has been reported in other ethnic groups. An abnormality (C1) of G to T substitution at cDNA 1376 causing an amino acid change from Arg to Leu has been found in 3 variants. Another abnormality (C2) of G to A substitution at cDNA 1388 causing an amino acid change from Arg to His has been found in 5 variants. Both C1 and C2 are located in exon 12 of the G6PD gene and are only 12 base pairs apart. However, C1 is associated with a significant increase in the deamino-NADP utilization rate, whereas C2 is not. Taken together, our data suggest that C1 and C2 are very common among Chinese with a G6PD deficiency and exon 12 may define an important functional domain of the human G6PD.

Base Sequence↗
PubMed1991

Prehemolytic effects of hydrogen peroxide and t-butylhydroperoxide on selected red cell properties.

To provide further understanding of how oxidative damage affects red cell membrane function, the effects of low levels of two different types of oxidants on selected red cell properties have been studied. Hydrogen peroxide (H2O2), an example of a water soluble oxidant, and t-butylhydroperoxide (tBHP), a hydrophobic hydroperoxide, were compared with respect to their effects on membrane permeability, membrane mechanical properties and binding of autologous serum antibodies to the cell surface. Whereas H2O2 treatment resulted in a dose-dependent increase in membrane permeability to potassium that was evident after one hour of oxidant exposure, cells treated with tBHP at doses up to 5 mumol/ml cells showed no immediate change in cation permeability. H2O2 also caused a marked decrease in membrane deformability, whereas tBHP-treated cells showed minimal loss of deformability. However, tBHP treatment did result in a dose-dependent increase in the susceptibility of the membrane to fragmentation under high shear stress. With exclusion of treated samples that bound excess rabbit anti-spectrin antibody, indicating exposure of intracellular components, neither agent promoted the binding of autologous serum antibody in amounts comparable to that found in vivo on high density or some pathologic red cells. Taken together, the results suggest that tBHP and H2O2 cause damage to human red cells by distinct oxidative mechanisms which do not lead directly to substantive generation of binding sites for autologous serum antibodies.

Carbon Dioxide↗
PubMed1991

NADPH, not glutathione, status modulates oxidant sensitivity in normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is characterized by the loss of NADPH and enhanced erythrocyte oxidant sensitivity. Historically, it has been theorized that the elevated oxidant sensitivity of G6PD-deficient erythrocytes arises as the direct consequence of decreased intracellular glutathione (GSH) concentrations. To directly investigate the basis of G6PD deficiency oxidant sensitivity, the effects of altered GSH and NADPH concentrations were examined in normal and G6PD-deficient erythrocytes. The results of this study demonstrated that GSH depletion, by 1-chloro-2,4-dinitrobenzene (CDNB), had no effect on hemoglobin oxidation in response to hydrogen peroxide (H2O2) generating systems (phenazine methosulfate and menadione bisulfite) in either normal or G6PD-deficient cells. Furthermore, a fourfold to sixfold increase in intracellular GSH concentration also did not protect against H2O2-generating systems in the normal or G6PD-deficient erythrocytes. Conversely, introduction of an NADPH-generating system (purified G6PD) into G6PD-deficient cells resulted in a significant decrease in oxidant sensitivity and an ability to cycle GSH. Further experiments demonstrated that the reduced oxidant sensitivity of the G6PD-reconstituted erythrocytes was not due to the maintenance of GSH levels because CDNB-mediated depletion of GSH did not alter this protective effect. Analysis of these results demonstrated a direct correlation between NADPH, but not GSH, concentration and hemoglobin oxidant sensitivity.

Dinitrochlorobenzene↗
PubMed1991

A comparative electrophysiological study on neurotisation in rats.

A comparative experimental study has been carried out in rats with denervated gastrocnemius muscles. Three groups of five rats were treated by three different types of implantation of nerve directly into muscle (neurotisation). In the fourth group, the common peroneal nerve was sutured to the transected tibial nerve. The fifth group was left denervated as a control. The muscles were studied by serial electrodiagnostic studies and later histologically. The results showed little difference between nerve suture and implantation of nerve directly into muscle. Extension of the common peroneal nerve with a pair of sural nerve grafts did not produce a detrimental effect.

Animals↗
PubMed1990

Transbilayer distribution and mobility of phosphatidylinositol in human red blood cells.

The present studies describe the distribution of phosphatidylinositol (PI) within the membrane bilayer of the human red blood cell (RBC) as well as its transbilayer mobility. The membrane bilayer distribution was determined by measuring the hydrolysis of PI in the exterior leaflet of the RBC membrane using a PI-specific phospholipase C and by extraction of PI from the exterior leaflet using bovine serum albumin. The transbilayer mobility of PI was measured by following the fate of radiolabeled PI which was first incorporated into the outer leaflet of the RBC membrane. Our results indicate that PI is asymmetrically distributed in the membrane, with approximately 80% located in the inner and 20% in the outer leaflet of the bilayer. The rate of transbilayer mobility of PI is similar to that for certain molecular species of phosphatidylcholine and much slower than that reported for the aminophospholipids in the RBC membrane.

Adult↗
PubMed1990

Excess of red cell membrane proteins in hereditary high-phosphatidylcholine hemolytic anemia.

Previous descriptions of hereditary high-phosphatidylcholine hemolytic anemia (HPCHA) have highlighted the association of increased erythrocyte membrane phosphatidylcholine with abnormal membrane cation permeability. We studied the function and composition of erythrocyte membranes from three individuals with HPCHA to characterize further the membrane abnormalities in this disorder. Despite significant macrocytosis, HPCHA red cells were dehydrated and showed an increased surface area to volume ratio compared to normal red cells. The passive efflux of K+ from HPCHA erythrocytes was increased fourfold at 37 degrees C. Total membrane phospholipid was increased 7-42%, largely due to excess phosphatidylcholine, which made up 35.8-37.2% of total phospholipid. Membrane cholesterol:phospholipid ratios were in the normal range. It appears that the excess phosphatidylcholine was not acquired during circulation, since plasma lipids were normal and all subpopulations of density-separated HPCHA erythrocytes were similarly abnormal. The ratio of total protein to phospholipid in white ghosts was increased, indicating that membrane protein was increased to an even greater extent than membrane lipids. No abnormal membrane proteins were identified by Coomassie or periodic acid Schiff (PAS) staining. Quantitation of the major membrane proteins indicated that the total protein excess in HPCHA membranes was due to a proportional increase in all major proteins. We conclude that HPCHA erythrocytes have excess membrane proteins and hypothesize that the changes in lipid composition and cation permeability are secondary to underlying protein abnormalities, which remain to be defined.

Adult↗
PubMed1990

The rectus abdominis muscle in difficult problems of hand soft tissue reconstruction.

When soft tissue losses in the hand require flap reconstruction, local tissue is preferred. Distant flaps should be reserved for major or unusually complex tissue losses which preclude the use of local flaps. We have used the rectus abdominis muscle as either a pedicled or a free flap with an overlying split thickness skin graft for several such complex soft tissue problems. Functional and cosmetic results have been excellent with minimal donor site morbidity. While not the initial choice for distant tissue used in hand reconstruction, the rectus abdominis muscle can solve several complex soft tissue problems in the hand.

Abdominal Muscles↗
PubMed1990

Transthecal digital block: flexor tendon sheath used for anesthetic infusion.

A new approach to achieve digital block by use of the flexor tendon sheath as an avenue introducing anesthetics to the core of the digit is described. Through centrifugal anesthetic diffusion all four digital nerves are anesthetized rapidly. This technique involves palmar percutaneous injection of 2 ml of lidocaine (Xylocaine) into the potential space of the flexor tendon sheath at the level of the palmar flexion crease with a 3 ml syringe and a No. 25-gauge hypodermic needle. Over the last 5 years, I have used this technique on 420 patients with no observable anesthetic complication. A repeat injection or local infiltration as a supplement was necessary only in four instances.

Fingers↗
PubMed1990

Rat ear reattachment as an animal model.

The external ear of the rat is an excellent model for practicing microsurgical dissection and for the refinement of microvascular anastomoses, techniques that are crucial for microvascular en bloc tissue transfer and replantation. Preparation of the rat ear for replantation requires familiarity with the vascular anatomy and gentle tissue handling with atraumatic dissection of arterial and venous pedicles, steps similarly crucial in raising free flaps for microvascular transfer. The strategy of performing accurate reduction and stabilization of the tubal cartilage prior to vessel repairs, anastomosing the more deeply seated external carotid artery prior to the more superficial posterior facial vein, is as critical to rat ear replantation as for digital reattachment. In addition, the rat ear as compared to other animal models such as the rabbit ear or canine hindlimbs is much less expensive. Compared to the rat hindlimb model, rat ears are much easier to observe, which is a distinct advantage when used as a model for long-term study of replantation, revascularization, or transplantation.

Animals↗
PubMed1990

A prospective clinical evaluation of autogenous vein grafts used as a nerve conduit for distal sensory nerve defects of 3 cm or less.

The purpose of this study was to determine the efficacy of autogenous vein grafts as nerve grafts (AVNC) for bridging of small peripheral sensory nerve gaps as compared with direct repair and with conventional nerve grafting techniques (ANG). Patients with painful neuroma or segmental nerve injury of 3 cm were chosen as the test group. Those amenable to direct repair were classified as controls. Between 1982 to 1988, a total of 22 patients were enrolled in this study. A total of 34 nerves were repaired, 15 with a venous nerve conduit, 4 with a sural nerve graft, and 15 with direct repair. Significant symptom relief and satisfactory sensory function return were uniformly observed. The two-point discrimination measurements indicated superiority of direct repair and probably of conventional nerve grafting. However, the universally favorable patient acceptance and the return of measurable two-point discrimination indicates the effectiveness of autogenous vein grafts as nerve conduits when selectively applied to bridge a small nerve gap (less than or equal to 3 cm) on nonessential peripheral sensory nerves.

Adult↗
PubMed1990

Use of ektacytometry to determine red cell susceptibility to oxidative stress.

To define a more sensitive and reliable method to determine changes in the overall cellular characteristics of erythrocytes after oxidative damage, we used a viscodiffractometric method (ektacytometry) to measure the effect of oxidative stress. Erythrocytes were incubated in the presence of hydrogen peroxide, t-butyl hydroperoxide, or cumene hydroperoxide in phosphate buffer. This treatment resulted in decreased cellular deformability of the intact erythrocytes. In addition, deformability and fragility measurements of the erythrocyte ghost membranes indicated an increased membrane dynamic rigidity and altered-mechanical stability as a consequence of oxidant stress. These changes were observed before the onset of hemolysis. The observed decrease in deformability was accompanied by oxidation of hemoglobin, alterations of membrane proteins, and lipid peroxidation. To continuously measure the time course of the decrease in deformability in intact erythrocytes under oxidative stress, a new ektacytometric method was developed. Erythrocytes were oxidatively challenged within the viscometer at a constant osmolality and shear stress. The change in deformability was monitored and a typical range was defined for erythrocytes from normal individuals. Comparison of erythrocytes from patients with sickle cell disease with those from normal individuals demonstrated a higher susceptibility of sickle red cells toward oxidative stress.

Erythrocyte Deformability↗
PubMed1989

Enhancement of erythrocyte superoxide dismutase activity: effects on cellular oxidant defense.

To delineate further the role of superoxide dismutase (SOD) in red blood cell (RBC) oxidant defense, normal human erythrocytes were osmotically lysed and resealed in the presence of varying concentrations of exogenous SOD. This resulted in a dose-dependent increase in SOD activity in the resealed erythrocytes while maintaining nearly normal RBC hemoglobin concentration (less than 10% decrease from the control value), cell volume, and cellular deformability. Surprisingly, a five- or ninefold increase in SOD activity yielded no additional protection against superoxide-generating drugs (phenazine methosulfate or menadione sodium bisulfite). No significant differences were observed between the control and SOD-loaded RBCs in O2-driven methemoglobin formation or generation of thiobarbituric acid-reactive substances. In contrast, RBCs with elevated SOD activity pretreated with sodium azide (to block catalase activity) or 1-chloro-2,4-dinitrobenzene (to deplete reduced glutathione, GSH) showed significantly enhanced methemoglobin generation in response to superoxide generating drugs. No differential response was noted between the control, control-resealed, and SOD-loaded RBCs to oxidants other than superoxide. Based on our results and other data, we conclude that elevated SOD activity may imbalance cellular oxidant defense, resulting in enhanced oxidation due to the accelerated generation of H2O2, the product of O2- dismutation. This effect is significantly exacerbated under conditions in which H2O2 catabolism is altered.

Antioxidants↗
PubMed1989

Chlorpromazine inhibits vesiculation, alters phosphoinositide turnover and changes deformability of ATP-depleted RBCs.

To delineate further the underlying mechanism by which amphiphilic drugs can modulate vesicle release from human RBCs, we studied the effect of chlorpromazine on erythrocyte vesiculation induced by ATP depletion. This was correlated with turnover of the phosphoinositides as well as RBC deformability during the process since phosphoinositide metabolism may be involved in shape regulation of RBCs. Echinocytic shape transformation and subsequent vesiculation of RBCs, which commonly occur during ATP depletion, were inhibited by chlorpromazine. Furthermore, with a newly developed two-dimensional thin-layer chromatography separation of RBC membrane phospholipids, we showed that chlorpromazine significantly decreased the dephosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) in both ATP-depleted RBCs as well as in cells with partly maintained ATP levels. Concomitantly, there was a smaller increase in the relative amount of phosphatidylinositol. In addition, chlorpromazine also inhibited the decreased in RBC deformability as well as the shift of osmotic fragility that occurs during ATP depletion of erythrocytes.

Adenosine Triphosphate↗
PubMed1989

Effects of neurite-promoting factors on rat sciatic nerve regeneration.

Neurite promoting factors (NPF), fibroblast growth factor, and laminin were instilled inside autologous venous nerve conduits (AVNC) interposed across a 1 cm gap in the rat sciatic nerve. Axonal regeneration was measured electrophysiologically and histologically and was compared to control AVNC and autologous nerve grafts (ANG) in 45 rats. By 1 month, the AVNC group with NPF recovered an average nerve conduction velocity twice that of the ANG group (24 m/sec vs. 12 m/sec). The gastrocnemius muscle action potential of NPF grafts was twice that of the control AVNC and was similar to the ANG (1.63 mV vs. 0.87 mV). In subsequent months, the advantage of NPF disappeared; these grafts were grossly reduced to fibrous bands. Histological examination of these grafts at 5 months revealed a florid intraluminal fibrovascular proliferation, which appeared to be "choking off" the regenerating axons. It is suggested that more refined mixtures of NPF should be instilled inside AVNC so that the improved axonal regeneration is maintained while the late inhibitory fibrosis is avoided.

Animals↗