The structure-function relationship of thrombin-like enzymes from the green pit viper (Trimeresurus albolabris).

Pit viper venoms can decrease fibrinogen levels in snakebite patients. Studies have shown that the hypofibrinogenemia is a consequence of snake venom thrombin-like enzymes (TLEs), the serine proteases that have the potential to be both diagnostic and therapeutic agents. Exosites of thrombin are the molecular regions that determine the substrate specificities, but its presence and significance in TLEs are unclear. Therefore, the putative exosites of recombinant TLEs derived from Green pit viper (Trimeresurus albolabris), GPV-TL1 and GPV-TL2, were mutated in a Pichia pastoris system. In a previous report, GPV-TL1 showed a strong fibrinogenolytic activity on the Aα and Bß chains of fibrinogen, as well as a plasma clotting activity. Compared with GPV-TL1, the GPV-TL1m mutated in the putative exosite (TRN to RRR at residues 60-62) showed a weaker fibrinogenolytic activity with a similar clotting activity of 207.1 thrombin units/mg. GPV-TL2 contained two-residue differences from GPV-TL1 in the putative exosite (N73M and V74Y). GPV-TL2 selectively cleaved only the Aα chain of fibrinogen without detectable clotting activity. The mutated GPV-TL2 (GPV-TL2m) showed a weaker fibrinogenolytic activity compared with that of the wild type. These results support the important roles of the putative exosite in snake venom TLE activities. This information is helpful for future protein engineering.

Analysis of green pit viper (Trimeresurus alborabris) venom protein by LC/MS-MS.

Green pit viper venom has major effect on the hematological system having a thrombin-like effect. Thus, this study is designed to analyze the composition of Trimeresurus albolabris venom by performing gel filtration and LC/MS-MS. The purified protein was then digested by trypsin, and the tryptic fragments were analyzed by iontrap spectrophotometry. This study found four types of proteins, namely jerdonitin, stejaggregin-A beta chain-1, stejnobin, and stejnihagin-A, as the components of T. albolabris venom. All of these toxins played a greater or lesser role in clot formation or otherwise contributed to cross-reactions in antivenom production.

Detection of Cryptococcus neoformans in bird excreta.

We evaluated 14 samples of bird excreta from pigeons, parrots, open billed storks and crows obtained from thirteen places in Bangkok and nearby areas between April and July 2004. These bird excreta were examined for Cryptococcus neoformans by direct plating method to inspect their ability to grow at 37 degrees C. Capsule production was examined by Indian ink preparation. They were also tested for urease and phenoloxidase enzymes. Cryptococcus neoformans var neoformans was recovered from pigeon excreta in 9.09%. This implies those having impaired immunity may get this fungus from the environment.

The effect of green pit viper (Trimeresurus albolabris) venom on platelet morphology by electron microscopy.

The incidence of venomous snake bites increases every year in Thailand, especially due to green pit viper. After the bite, there is bleeding due to thrombin-like property of the venom. The mean platelet volume has been reported to be decreased in those who have been bitten by this snake. In this study we investigate the effect of green pit viper venom (Trimeresurus albolabris) on platelet volume (MPV), number and morphology of platelets in vitro. The test was carried out by washing platelets in phosphate buffer at pH 7.2 to remove fibrinogen, then the washed platelets were mixed with green pit viper venom. Platelet morphology was examined by scanning electron microscope (SEM). The morphology of platelets was smaller than normal which ranges from 1.1- 1.2 microm. Green pit viper venom can directly effect platelet morphology, decreasing platelet volume.

Preliminary study of the effect of vitamin E supplementation on the antioxidant status of hemoglobin-E carriers.

The antioxidant status of hemoglobin-E carriers was studied pre- and post-treatment with vitamin E for 3 months. Fourteen hemoglobin-E carriers (age = 21.36 +/- 1.08 years, BMI = 18.32 +/- 1.22 kg/m2) were treated with vitamin E 200 I.U. daily for 3 months. Fasting blood samples were collected and analyzed for erythrocyte superoxide dismutase activity, total antioxidant activity, hemoglobin concentration, hematocrit, MCV, Heinz body formation and osmotic fragility test. The blood parameters before and after vitamin E treatment were compared. The results showed that superoxide dismutase activity in the erythrocytes was significantly decreased, while total antioxidant activity in plasma, and the osmotic fragility of the erythrocytes, was significantly increased after vitamin E supplementation. However, hematocrit, MCV, and Heinz body formation did not change significantly. This demonstrated that vitamin E 200 IU could be used as a lipophilic antioxidant in red blood cells and could help increase the level of antioxidant in hemoglobin-E carriers.

Inhibition of Heinz body induction in an in vitro model and total antioxidant activity of medicinal Thai plants.

Herbs have been used for medicinal purposes for centuries and known to possess antioxidant properties that may help to reduce the risk of chronic diseases, cardiovascular disease, and cancer. We screen aqueous extracts from 20 medicinal plants in Thailand that were believed to possess anti-tumor activity, help immune-stimulating property and maintain blood stasis. The antioxidant activities were investigated in two bioassays. Firstly, we demonstrated inhibition of Heinz bodies induction caused by oxidants under in vitro condition. The percentages of Heinz body inhibition activity in plant extracts from Terminalia citrina, Cassia timoriensis, and Derris elliptica were the highest followed by Anamirta cocculus, and Oroxylum indicum respectively. In addition, we investigated total antioxidant activity in plant extracts by improved ABTS radical cation decolorization assay. The total antioxidant activity of the extract from Terminalia citrina was also the highest activity followed by Ficus pubigera, Derris elliptica, Anamirta cocculus, Caesalpinia sappan, and Oroxylum indicum respectively. Our results suggest medicinal Thai plants as valuable sources of antioxidants, which may have a potential anti-carcinogenic activity.

Antioxidant in plasma of hemoglobin-E trait.

A study of antioxidant levels among Thai subjects with a hemoglobin E trait was undertaken. The objective of this study was to determine whether the antioxidant level would be disturbed in the HbE condition. All 185 volunteer subjects, 171 normal healthy subjects and 14 HbE carriers were recruited. For each case, an antioxidant determination was performed using the Trolox equivalent antioxidant capacity (TEAC) method. According to this study, the average antioxidant level in the healthy group was 3.439 +/- 0.220 mM Trolox equivalent, and in HbE trait group was 3.276 +/- 0.209 mM Trolox equivalent. There was a significant decrease of the antioxidant level in the HbE trait group (p = 0.008).

Platelet count and platelet parameters in hemoglobin E carriers.

Hemoglobinopathies are an important inherited disorder with a high prevalence in Thailand. Of several hemoglobinopathies, hemoglobin E (Hb) disorder (beta 26, GAG-AAG, Glu-Lys) is the most common. Coagulation disorders in these patients have also been proposed. Even though thrombotic risks in the patients with hemoglobin disorders from standpoints of platelet dysfunction and coagulation factors are controversial, they are in favor of thrombosis due to thrombocytosis. A study was performed in 57 healthy subjects to evaluate platelet count and platelet parameters in hemoglobin E carriers compared to values in healthy subjects. Classified by standard hemoglobin electrophoresis, there were 46 healthy subjects and 11 hemoglobin E carriers. There are no significant differences in platelet count and platelet parameters between the two groups (p > 0.05). Although there are reports that indicate the change in the quality of platelet in hemoglobin E disorders, no quantitative disorder was detected. There was no trend toward increased platelet count in the HbE carriers.