Wheat bran protects Fischer-344 rats from diquat-induced oxidative stress by activating antioxidant system: selenium as an antioxidant

Wheat bran had a protective effect against diquat toxicity in rats fed a purified diet (PD). We studied the effects of wheat bran on the antioxidant system in the liver of rats treated with saline and diquat. Although feeding wheat bran did not affect the concentration of hepatic non-protein sulfhydryl or the activity of glucose 6-phosphate dehydrogenase in the saline-injected rats, these values were significantly higher in the rats fed PD containing wheat bran (W-PD) than in rats fed only PD after administering diquat. The glutathione peroxidase and reductase activities were significantly elevated by wheat bran in the saline-injected rats. Although the glutathione peroxidase activity was unchanged in both the PD-fed rats and W-PD-fed rats after the diquat treatment, the glutathione reductase activity was significantly decreased in both the PD-fed and W-PD-fed rats. Feeding the rats with PD containing 0.15 ppm selenium as well as with W-PD elevated the activity of hepatic glutathione peroxidase and attenuated the diquat toxicity. These results indicate that wheat bran protected against diquat toxicity by activating the hepatic antioxidant system, and that selenium was the key antioxidant in wheat bran.     

Effects of zinc and cadmium on erythrocyte antioxidant systems of a freshwater fish Oreochromis niloticus

In this work to determine the effects of metals exposure of Oreochromis niloticus on erythrocyte antioxidant systems, fish were exposed to 5.0 mg/L Zn, 1.0 mg/L Cd, and 5.0 mg/L Zn + 1.0 mg/L Cd mixtures for 7 and 14 days and reduced glutathione (GSH) level, catalase (CAT), and glucose‐6‐phosphate dehydrogenase (G6PD) activities were investigated. In addition, Zn or Cd levels in whole blood were studied. Erythrocyte GSH level and CAT and G6PD enzyme activities increased in response to single and combined Zn and Cd exposure. The elevation observed in the CAT activity was higher in the Cd alone, and in combination with Zn, than in Zn alone. Time‐dependent alteration was not observed in all antioxidant parameters. Exposure to metals (alone and in mixture) resulted in elevatation of Zn and Cd levels in the blood. Concentration of metals in the blood of fish exposed to the Zn + Cd combination was lower than in fish exposed to the single metal. This study demonstrates that metals caused oxidative stress in fish erythrocytes, and an adaptation with an increase in CAT and G6PD activities and GSH level, which were important in the protection against metal damage, was observed. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:223–229, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20327     

Examination of Changes in Enzyme Activities of Erythrocyte Glucose 6‐Phosphate Dehydrogenase and 6‐Phosphogluconate Dehydrogenase in Rats Given Naringenin and Lead Acetate

In our study, controlled experimental groups were performed by giving substances Lead acetate, Naringenin and Naringenin + Lead acetate to rats in vivo conditions Changes in the glucose 6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzyme activities in erythrocytes of rats in these groups were compared to the Control group. An inhibition significant degree for G6PD enzyme activity was observed in all groups when compared to the Control group (p < 0.001). While inhibition significant degree for 6PGD enzyme activity was observed in Lead acetate groups (p < 0.001), no significant effect was observed in the Naringenin and Naringenin + Lead acetate groups (p > 0.05). In addition, lead levels in the groups of rats were determined using an inductively coupled plasma mass spectrometer (ICP‐MS) device. As a result of measurements by the ICP‐MS device, lead levels were found as an average of 42.9 ± 2.51, 36.71 ± 1.13, 172.16 ± 9.63, and 95.07 ± 5.87 ppm in the Control, Naringenin, Lead acetate and Naringenin + Lead acetate groups, respectively. Our results were shown that Naringenin has protective effects on the Lead acetate induced oxidative stress erythrocytes in rat.     

Combined effects of 2,4-D and azinphosmethyl on antioxidant enzymes and lipid peroxidation in liver of Oreochromis niloticus

This study aims to investigate the effects of the herbicide 2,4-D and the insecticide azinphosmethyl on hepatic antioxidant enzyme activities and lipid peroxidation in tilapia. Fish were exposed to 27 ppm 2,4-D, 0.03 ppm azinphosmethyl and to a mixture of both for 24, 48, 72 and 96 h. Activities of catalase (EC 1.11.1.6), glutathione-S-transferase (GST, EC 2.5.1.18) and the level of malondialdehyde (MDA) in the liver of Oreochromis niloticus exposed to 2,4-D and azinphosmethyl, both individually and in combination, were not affected by the pesticide exposures. However, glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) and glutathione reductase (GR, EC 1.6.4.2) activities in individual and combined treatments, increased significantly compared to controls. Furthermore, glutathione peroxidase (GPx, EC 1.11.1.9) activity increased in individual treatment, while the same enzyme activity decreased in combination. 2,4-D did not affect the activity of superoxide dismutase (SOD, EC 1.15.1.1), but the activity of this enzyme in azinphosmethyl treatment decreased, while its activity increased in combination. Combined treatment of the pesticides exerted synergistic effects in the activity of SOD, while antagonistic effects were found in the activities of G6PD, GPx, GR. The results indicate that O. niloticus resisted oxidative stress by antioxidant mechanisms and prevented increases in lipid peroxidation.     

Purification and properties of glucose 6-phosphate dehydrogenase from turkey erythrocytes

Glucose 6-phosphate dehydrogenase (G6PD) was purified from turkey erythrocytes by ammonium sulphate precipitation and followed by ADP Sepharose affinity gel chromatography. The yield was 49.71% and specific activity of the enzyme was found to be 44.16 EU/mg protein. By gel filtration the molecular mass was found to be 75 kDa. The enzyme had an optimum pH at 9.0, and optimum temperature at 50 degrees C. Km and Vmax for NADP(+) and glucose 6- phosphate (G6-P) as substrates were also determined and effects of inhibitors such as ATP, NADH and NADPH were examined.     

The effect of astaxanthin and cadmium on rat erythrocyte G6PD, 6PGD,GR, and TrxR enzymes activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro

In this study, the effects of astaxanthin (AST) that belongs to carotenoid family and cadmium (Cd), which is an important heavy metal, on rat erythrocyte G6PD, 6PGD, GR, and TrxR enzyme activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro were studied. In in vitro studies, 6PGD enzyme was purified from rat erythrocytes with 2′,5′‐ADP Sepharose4B affinity chromatography. Results showed inhibition of enzyme by Cd at IC₅₀; 346.5 μM value and increase of 6PGD enzyme activity by AST. In vivo studies showed an increase in G6PD, 6PGD, and GR enzyme activities (P ? 0.05) and no chance in TrxR enzyme activity by AST. Cd ion inhibited G6PD, 6PGD, and GR enzyme activities (P ? 0.05) and also decreased TrxR enzyme activity (P ? 0.05). AST + Cd group G6PD enzyme activity was statistically low compared with control group (P ? 0.05). 6PGD and TrxR enzyme activities decreased without statistical significance (P ? 0.05); however, GR enzyme activity increased statistically significantly (P ? 0.05).     

Effect of astaxanthin and aluminum chloride on erythrocyte G6PD and 6PGD enzyme activities in vivo and on erythrocyte G6PD in vitro in rats

In this study, we investigated the effect of astaxanthin (Ast) and aluminum (Al) on the erythrocyte glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activities in vivo and on G6PD enzyme in vitro in rats. For in vitro studies, G6PD enzyme was purified from rat erythrocyte by using 2′,5′‐ADP‐Sepharose 4B affinity gel. The effects of Ast and Al³⁺ ion were investigated on the purified enzyme. It was determined that Ast increased the enzyme activity, whereas Al³⁺ inhibited the enzyme activity noncompetitively (IC₅₀ values; 0.679 mM, K_i values 1.32 mM). For in vivo studies, the rats were divided into the groups: control (Cont.), Al, Ast, and Al + Ast. The last three groups were compared with the control group. In Al group, a significant degree of inhibition was observed in the activity of G6PD and 6PGD enzymes when compared with the control group (P < 0.05), whereas there was an increase in the activities of G6PD and 6PGD enzymes in Ast and Al + Ast groups (P < 0.05).     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

Sublethal ammonia and urea concentrations inhibit rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase

In vitro and in vivo effects of sublethal ammonia and urea concentrations were assayed on glucose-6-phosphate dehydrogenase (G6PD) of rainbow trout (Oncorhynchus mykiss) erythrocyte. G6PD was purified from erythrocytes with a specific activity of 16.7 EU (mmol NADP+/min)/mg protein and approximately 1600-fold in a yield of approximately 60% by ammonium sulphate precipitation and 2',5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was confirmed using SDS polyacrylamide gel electrophoresis. Experiments with ammonia (2.2-5.5 microM) and urea (20-50 microM) showed the inhibitory effects on the enzyme, in vitro. Inhibition effects were determined in vitro by Lineweaver-Burk and regression graphs. The dissociation constant of the enzyme inhibitor complex (Ki) and 50% inhibitory values were 2.26+/-1.21 and 2.86+/-3.51 microM for ammonia and 18.69+/-6.75 and 23.77+/-4.58 microM for urea, respectively. In vivo studies in rainbow trout erythrocytes showed significant (p < 0.01) inhibition of G6PD by ammonia and urea. However, ammonia inhibited more than urea since there were significant differences between the final values of erythrocyte G6PD activities.     

Glucose-6-Phosphate Dehydrogenase Activity and Glutathione Stability in Fetal and Adult Bovine Erythrocytes

Previously, we found a substantially higher glucoses-phosphate dehydrogenase (G6PD) activity and a slightly higher 6-phosphogluconate dehydrogenase (6PGD) activity in bovine fetal erythrocytes than in bovine adult erythrocytes (Steensgaard 1968). Now, we have investigated whether these differences in dehydrogenase activities were followed by characteristic differences in glutathione (GSH) stability and glutathione concentration. The results are shown in Table 1, which also gives the results of the same investigations on normal and G6PD deficient human erythrocytes.     

Postnatal Expression of Glucose-6-Phosphate Dehydrogenase in Different Brain Areas

The activity of glucose-6-phosphate dehydrogenase (G6PD) was studied in five brain areas of rats aged 5 to 90 days. The areas studied were: the olfactory bulb (OB), cortex, hippocampus, striatum and septum. The G6PD activity increased more than 2-fold from 5 to 90 days in the OB, while it was almost constant in the other areas. At every stage of development, the G6PD activity was significantly higher in the OB than in the other areas. The G6PD pattern was compared with 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR); glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) in order to find synergistic interactions among activities of these enzymes during development. Over the considered period, the activity of 6PGD increased significantly in the OB, while no significant difference in activity was detected in the other areas. GR increased significantly and progressively at each developmental stage in all areas. GPX showed a progressive increase in the OB, while in other areas a significant increase was detected at 90 days only. CAT and SOD showed a different and independent pattern which differred from the G6PD pattern. CAT showed the highest level of activity at 5 days then progressively decreased or was constant until 90 days; SOD had the highest value at 5 days, than it decreased at 10 days and increased from 10 to 90 days. In all areas, G6PD activity showed three electrophoretic bands, whose relative activity changed with development. At histochemical level, we found a marked G6PD activity in the periglomerular zone of the OB, which increased with age, while other areas showed a homogeneous staining. The present results demonstrate that G6PD activity increases in the OB during the developmental stages and there is a coordinated simultaneous activation of 6PGD, GPX and GR. It is likely that this enzyme induction increases the antioxidant defense of periglomerular cells that are subject to a rapid renewal and thus much more exposed to oxidant stress.     

Purification of glucose 6-phosphate dehydrogenase from chicken erythrocytes. investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from chicken erythrocytes, and some characteristics of the enzyme were investigated. The purification procedure was composed of three steps: hemolysate preparation, ammonium sulfate precipitation, and 2',5'-ADP Sepharose 4B affinity gel chromatography. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 20.862 EU/mg proteins, was purified with a yield of 54.68% and 9,150-fold. Optimal pH, stable pH, optimal temperature, molecular weight, and KM and Vmax values for NADP+ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, Ki values and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADH, and NADPH.     

Erythrocyte antioxidant enzymes in hypertensive patients receiving lisinopril monotherapy or combined lisinopril plus simvastatin therapy

Statins and angiotensin-converting enzyme (ACE) inhibitors have beneficial impact on the serum cholesterol and blood pressure. It is supposed that statins and ACE inhibitors may modify the antioxidative status in erythrocytes. The study objective was to compare the effects of two treatments, lisinopril alone versus lisinopril plus simvastatin, on erythrocyte antioxidant enzyme activities. The study involved 32 patients with arterial hypertension, their initial serum total cholesterol, LDL-cholesterol and triglycerides were within the normal range. Patients of two groups, each of 16 subjects, were treated with lisinopril (10 mg/day) or with lisinopril (10 mg/day) plus simvastatin (20 mg/day). Before and after the ambulatory therapy for 3 and 6 months, activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR) were determined in purified erythrocytes. All treated patients had significantly higher catalase activity (by 79.3–106.5%, p < 0.0001) and significantly lower GPx activity (by 20.7–30.6%, p < 0.001) as compared to the baselines. The same results were obtained in both groups (lisinopril and lisinopril + simvastatin), after both periods (3 and 6 month) of treatments. SOD activity increased only in the lisinopril group and only after 6 months (p = 0.0345). No changes of GR activity were observed under all conditions studied. Thus, the lisinopril monotherapy and combined lisinopril plus simvastatin therapy exhibit specific, pronounced and equipotent effects on antioxidant enzymes in human erythrocytes. Peroral administration of lisinopril or lisinopril plus simvastatin may protect erythrocytes and other tissues against oxidative damage.     

Glucose-6-phosphate dehydrogenase in rat lung alveolar epithelial cells. An ultrastructural enzyme-cytochemical study

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway in carbohydrate metabolism, and it plays an important role in cell proliferation and antioxidant regulation within cells in various organs. Although marked cell proliferation and oxidant/antioxidant metabolism occur in lung alveolar epithelial cells, definite data has been lacking as to whether cytochemically detectable G6PD is present in alveolar epithelial cells. The distribution pattern of G6PD within these cells, if it is present, is also unknown. The purpose of the present study was to investigate the subcellular localization of G6PD in alveolar cells in the rat lung using a newly-developed enzyme-cytochemistry (copper-ferrocyanide) method. Type I cells and stromal endothelia and fibroblasts showed no activities. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of type II alveolar epithelial cells. The cytochemical controls ensured specific detection of enzyme activity. This enzyme may play a role in airway defense by delivering substances for cell proliferation and antioxidant forces, thus maintaining the airway architecture.     

Cyclosporine A induced changes to plasma and erythrocyte antioxidant defences

Organ transplant recipients develop pronounced cardiovascular disease, and decreased antioxidant capacity in plasma and erythrocytes is associated with the pathogenesis of this disease. These experiments tested the hypothesis that the immunosuppressant cyclosporine A (CsA) alters erythrocyte redox balance and reduces plasma antioxidant capacity. Female Sprague-Dawley rats were randomly assigned to a control or CsA treated group. Treatment animals received 25 mg/kg/day of CsA via intraperitoneal injection for 18 days. Control rats were injected with the same volume of the vehicle. Three hours after the final CsA injection, rats were exsanguinated and plasma analysed for total antioxidant status (TAS), alpha-tocopherol, malondialdehyde (MDA), and creatinine. Erythrocytes were analysed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities, alpha-tocopherol, and MDA. CsA administration resulted in a significant (P < 0.05) decrease in plasma TAS and significant increases (P < 0.05) in plasma creatinine and MDA. Erythrocyte CAT was significantly (P < 0.05) increased in CsA treated rats compared to controls. There were no significant differences (P > 0.05) in erythrocyte SOD, GPX, G6PD, alpha-tocopherol or MDA between groups. In summary, CsA alters erythrocyte antioxidant defence and decreases plasma total antioxidant capacity.     

Seasonal changes in antioxidant defence system of liver and gills of Salmo trutta caspius, Salmo trutta labrax and Salmo trutta macrostigma

Seasonal changes in antioxidant enzyme activities (superoxide dismutase, SOD, EC 1.15.1.1; catalase, CAT, EC 1.11.1.16; glutathione peroxidase, GPx, EC 1.11.1.9; glutathione reductase, GR, EC 1.6.4.2; glucose-6-phosphate dehydrogenase, G6PD, EC 1.1.1.49 and glutathione S -transferase, GST, EC 1.5.1.18) and lipid peroxidation (LPO) levels of livers and gills of female Caspian trout Salmo trutta caspius , Black Sea trout Salmo trutta labrax and mountain trout Salmo trutta macrostigma were investigated. SOD, CAT, GPx, G6PD and GST activities were higher in liver compared to gills of all sub-species; concomitantly, the GR activity was also higher in the livers of S. t. caspius and S. t. labrax , but the reverse was seen in S. t. macrostigma . LPO levels were higher in the gills compared to the liver of all sub-species. There was no general trend in the seasonal changes in the gill antioxidant enzyme (AE) activities or LPO levels. Higher AE activities, however, were found in the liver of each sub-species during autumn, and this coincided with an increase in the gonado-somatic index.     

Oxidative stress correction by nicotinamide and nicotynol-GABA in diabetic neuropathy

Concentration of lipid peroxidation products and antioxidant enzyme activities in rat brain and erythrocytes and the effects of nicotinamide and nicotinoyl-GABA administration on these parameters were estimated on 21st day of streptozotocin-induced diabetes. It was demonstrated more then two-fold diabetes-induced accumulation of conjugated dienes and malondialdehyde in tissues studied. Superoxide dismutase and glutathione reductase activities of both brain homogenate and erythrocytes as well as catalase and glutathione peroxidase activities of brain homogenate were shown to decrease significantly in diabetic rats, meanwhile, catalase activity of erythrocytes was increased and glutathione peroxidase unchanged. So the correlation between changes in enzymatic antioxidant system in brain and erythocytes failed to be found. Alterations observed were virtually prevented by the course of nicotinamide and nicotinoyl-GABA treatment. The results suggested that the suppression of antioxidant system could be primary biochemical disturbance in diabetic neuropathy progression. It was shown that the antioxidant efficacy of nicotinoyl-GABA is lower than that of nicotinamide. It was suggested that the mechanism of antioxidant action of nicotinamide and its structural analogue consists of both scavenging of lipid peroxides and NAD biosynthesis that leads to activation and normalization of altered energy and lipid metabolism.     

Crocidolite asbestos inhibits pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human lung epithelial cells

The cytotoxicity of asbestos has been related to its ability to increase the production of reactive oxygen species (ROS), via the iron-catalyzed reduction of oxygen and/or the activation of NADPH oxidase. The pentose phosphate pathway (PPP) is generally activated by the cell exposure to oxidant molecules. Contrary to our expectations, asbestos (crocidolite) fibers caused a dose- and time-dependent inhibition of PPP and decreased its activation by an oxidative stress in human lung epithelial cells A549. In parallel, the intracellular activity of the PPP rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD), was significantly diminished by crocidolite exposure. This inhibition was selective, as the activity of other PPP and glycolysis enzymes was not modified, and was not attributable to a decreased expression of G6PD. On the opposite, the incubation with glass fibers MMVF10 did not modify PPP and G6PD activity. PPP and G6PD inhibition did not correlate with the increased nitric oxide (NO) production elicited by crocidolite in A549 cells. Experiments with the purified enzyme suggest that crocidolite inhibits G6PD by directly interacting with the protein. We propose here a new mechanism of asbestos-evoked oxidative stress, wherein fibers increase the intracellular ROS levels also by inhibiting the main antioxidant pathway of the cell.     

Metabolic responses to prolonged starvation, food restriction, and refeeding in the brown trout, Salmo trutta: oxidative stress and antioxidant defenses

The effects of long-term starvation and food restriction (49 days), followed by refeeding (21 days) have been studied with respect to antioxidant defense in the liver and gills (branchial tissues) of the brown trout, Salmo trutta. Malondialdehyde levels in both tissues increased in parallel with starvation and food restriction and these values did not return to normal after the refeeding period. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in liver and gills increased during the 49 days of starvation, but glucose-6-phosphate dehydrogenase (G6PD) activities decreased. Glutathione S-transferase (GST) activity decreased in the liver at the 49th day of starvation, but increased in the branchial tissues. Some of the antioxidant enzyme activities (such as hepatic GST and branchial G6PD) returned to control values of fed fish after the refeeding period, but others (e.g. hepatic SOD and branchial GPx) did not return to normal values. In conclusion, our study indicates that total or partial food deprivation induces oxidative stress in brown trout.     

Glucose-6-phosphate dehydrogenase – from oxidative stress to cellular functions and degenerative diseases

Abstract

Glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the pentose phosphate pathway, is indispensable to maintenance of the cytosolic pool of NADPH and thus the cellular redox balance. The role of G6PD as an antioxidant enzyme has been recognized in erythrocytes for a long time, as its deficiency is associated with neonatal jaundice, drug- or infection-mediated hemolytic crisis, favism and, less commonly, chronic non-spherocytic hemolytic anemia. To a large extent, advances in the field were made on the pathophysiology of G6PD-deficient erythrocytes, and the molecular characterization of different G6PD variants. Not until recently did numerous studies cast light on the importance of G6PD in other aspects of the physiology of both cells and organisms. Deficiency in G6PD activity, and hence a disturbance in redox homeostasis, can lead to dysregulation of cell growth and signaling, anomalous embryonic development, altered susceptibility to viral infection as well as increased susceptibility to degenerative diseases. The present review covers recent developments in this field. Additionally, molecular characterization of G6PD variants, especially those frequently found in Taiwan and Southern China, is also addressed.