Biodistribution, pharmacokinetics and toxicity of a Vasconcellea cundinamarcensis proteinase fraction with pharmacological activity

Abstract Prior studies demonstrate that a proteinase fraction from Vasconcellea cundinamarcensis V.M. Badillo, Caricaceae, exhibits wound healing activity in gastric and cutaneous models and antitumoral/antimetastatic effects. Here, we present the toxicity, pharmacokinetics and biodistribution data for this proteinase fraction following a single dose into Swiss mice by i.v., s.c. or p.o. routes. The i.v. and s.c. toxicity assays demonstrate that proteinase fraction at ≤20 mg/kg is non-lethal after single injection, while parental administration (p.o.) of ≤300 mg/kg does not cause death. Based on p.o. acute toxicity dose using Organisation for Economic Cooperation and Development protocols, proteinase fraction ranks as Class IV “harmful” substance. Proteinase fraction shows high uptake determined as Kp (distribution tissue/blood) in organs linked to metabolism and excretion. Also, high bioavailability (≈100%) was observed by s.c. administration. The blood contents following i.v. dose fits into a pharmacokinetic bi-compartmental model, consisting of high removal constants – kel 0.22 h−1 and kd 2.32 h−1and a half-life – t½ = 3.13 h. The Ames test of proteinase fraction (0.01–1%) demonstrates absence of mutagenic activity. Likewise, genotoxic evaluation of proteinase fraction (5 or 10 mg/kg, i.p.) shows no influence in micronuclei frequency. In conclusion, the acute doses for proteinase fraction lack mutagenic and genotoxic activity, clearing the way for clinical assays.

Potential of laticifer fluids for inhibiting Aedes aegypti larval development: evidence for the involvement of proteolytic activity

It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.

Cloning Expression and Characterization of Two Cysteine Proteinases of Clonorchis sinensis / 热带医学杂志

Objective To clone and express the valuable Clonorchis sinensis antigen molecules which can be applied to the diagnosis of clonorchiasis. Methods Based on the sequences (Genbank) No. AF271091 (CysA) and No.AF093242 (CysB), primers were designed to amplify the two C. sinensis cysteine proteinase genes and expressed in E.cloi. The expressed proteins were purified by affinity chromatography and then tested for their immunological characters.Results The two genes were successfully cloned and expressed. Western blot showed that CysB had strong reaction with clonorchiasis sera and very weak reaction with schistosomiasis sera, while CysA showed no reactivity with the probed sera. Immunohistochemistry showed that both proteins were mainly located in adult worm intestines and the intrauterine eggs.Conclusions The results suggested that, of the two expressed C. sinensis proteins, CysB had good antigenic reactivity against sera from patients. It is a potential candidate of diagnostic antigens for clonorchiasis.

Expression of Survivin and Caspase-3 protein and their relationships with apoptosis in colorectal carcinoma / 中国医师杂志

Objective To evaluate the expressions of Survivin and Caspase-3 in patients with colorectal carcinoma and their association with tumor cell apoptosis.Methods The TdT-mediated dUTP nick end labelling(TUNEL) and immunohistochemistry were used to study cell apoptosis and expression of Survivin and Caspase-3 in 68 cases of colorectal cancer tissues and 20 normal controls.Results Survivin was expressed in 36 of 68(52.9%) cases of colorectal carcinoma and was not expressed in normal tissues.The expression of Survivin was significantly correlated with lymph node metastasis and histological type(P

Effect of hypoxia ischemia on cysteinyl aspartate specific proteinases activity in cerebral tissue of neonatal rat / 中华妇产科杂志

Objective To study the effect of hypoxia ischemia on cysteinyl aspartate specific proteinases (caspase 3) activity in cerebral tissue of neonatal rat and probe into its significance Methods To induce hypoxic ischemic brain damage (HIBD), the left carotid artery of rats at day 7 was ligated and animals were exposed to 8% oxygen for 2 hours 0 5,12,24,and 48 hours after HIBD, both ipsilateral and contralateral cerebral tissue were ditected and homogenized caspase 3 activity was measured by cleavage of the colorimetric substrate DEVD pNA Results Caspase 3 activity in ipsilateral cerebral tissue increased gradually after HIBD and peaked at 24 hours, and then decreased significantly at 48 hours( P 0 05) Conclusions Significant activation of caspase 3 after cerebral hypoxia ischemia strongly suggests that apoptosis is involved in HIBD Application of caspase inhibitors or other anti apoptotic agents may become a new therapeutics of HIBD