Agaritine derived from Agaricus blazei Murrill induces apoptosis via mitochondrial membrane depolarization in hematological tumor cell lines.

Objectives: Agaritine (AGT) is a hydrazine-containing compound derived from the mushroom Agaricus blazei Murill. We previously reported the antitumor effect of AGT on hematological tumor cell lines and suggested that AGT induces apoptosis in U937 cells via caspase activation. However, the antitumor mechanism of AGT has not been fully understood. Methods: Four hematological tumor cell lines (K562, HL60, THP-1, H929) were used in this study. The cells were incubated in the presence of 50 µM AGT for 24 h and analyzed for cell viability, annexin V positivity, caspase-3/7 activity, mitochondrial membrane depolarization, cell cycle, DNA fragmentation, and the expression of mitochondrial membrane-associated proteins (Bax and cytochrome c). Results: In HL60, K562, and H929 cells, AGT reduced cell viability and increased annexin V- and dead cell-positive rates; however, it did not affect THP-1 cells. In K562 and HL60 cells, caspase-3/7 activity, mitochondrial membrane depolarization, and expression of mitochondrial membrane proteins, Bax and cytochrome c, were all increased by AGT. Cell cycle analysis showed that only K562 exhibited an increase in the proportion of cells in G2/M phase after the addition of AGT. DNA fragmentation was also observed after the addition of AGT. Conclusions: These results indicate that AGT induces apoptosis in K562 and HL60 cells, like U937 reported previously, but showed no effect on THP-1 cells. It was suggested that AGT-induced apoptosis involves the expression of Bax and cytochrome c via mitochondrial membrane depolarization.

Evaluation of a new method for the diagnosis of alterations of Lens culinaris agglutinin binding of thyroglobulin molecules in thyroid carcinoma.

BACKGROUND: The measurement of serum thyroglobulin (Tg) is widely used as a marker for recurrence of thyroid carcinoma following total thyroidectomy. However, this method cannot differentiate between benign and malignant disease. We focused on the sugar chain in the Tg molecule and investigated the usefulness of Lens culinaris agglutinin (LCA)-reactive Tg ratios in sera and wash fluids obtained during fine-needle aspiration (FNA) for the detection of thyroid carcinoma. METHODS: The study was performed using 203 serum samples (115 from patients with benign thyroid disease and 88 from patients with thyroid carcinomas) and 176 wash fluid samples (143 benign, 21 malignant, and 12 inconclusive). LCA-reactive Tg ratios were determined using an enzyme-linked immunosorbent assay, and a comparison was made between malignant and benign lesions. RESULTS: In serum, the ratio in patients with malignancy was 79.5+/-6.0 [mean+/-standard deviation (SD)], significantly lower than in patients with benign lesions (84.9+/-3.5). The ratios in wash fluid from malignant lesions (75.8+/-18.9) were also significantly lower than those from benign lesions (85.6+/-3.9). CONCLUSIONS: These results suggest that this method could distinguish between benign and malignant lesions and may be useful for screening serum and wash samples.

Implementation of a novel PCR based method for detecting malaria parasites from naturally infected mosquitoes in Papua New Guinea.

BACKGROUND: Detection of Plasmodium species in mosquitoes is important for designing vector control studies. However, most of the PCR-based detection methods show some potential limitations. The objective of this study was to introduce an effective PCR-based method for detecting Plasmodium vivax and Plasmodium falciparum from the field-caught mosquitoes of Papua New Guinea. METHODS: A method has been developed to concurrently detect mitochondrial cytochrome b (Cyt b) of four human Plasmodium species using PCR (Cytb-PCR). To particularly discriminate P. falciparum from P. vivax, Plasmodium ovale and Plasmodium malariae, a polymerase chain reaction-repeated fragment length polymorphism (PCR-RFLP) has further been developed to use with this method. However, due to limited samples number of P. ovale and P. malariae; this study was mainly confined to P. vivax and P. falciparum. The efficiency of Cytb-PCR was evaluated by comparing it with two 'gold standards' enzyme linked immunosorbent assay specific for circumsporozoite protein (CS-ELISA) using artificially infected mosquitoes; and nested PCR specific for small subunit ribosomal RNA (SSUrRNA) using field caught mosquitoes collected from three areas (Kaboibus, Wingei, and Jawia) of the East Sepic Province of Papua New Guinea. RESULTS: A total of 90 mosquitoes were artificially infected with three strains of Plasmodium: P. vivax-210 (n = 30), P. vivax-247 (n = 30) and P. falciparum (n = 30). These infected mosquitoes along with another 32 unfed mosquitoes were first checked for the presence of Plasmodium infection by CS-ELISA, and later the same samples were compared with the Cytb-PCR. CS-ELISA for P. vivax-210, P. vivax-247 and P. falciparum detected positive infection in 30, 19 and 18 mosquitoes respectively; whereas Cytb-PCR detected 27, 16 and 16 infections, respectively. The comparison revealed a close agreement between the two assays (kappa = 0.862, 0.842 and 0.894, respectively for Pv-210, Pv-247 and P. falciparum groups). It was found that the eight CS-ELISA-positive mosquitoes detected negative by Cytb-PCR were false-positive results. The lowest detection limit of this Cytb-PCR was 10 sporozoites. A highly concordance result was also found between nested PCR and Cytb-PCR using 107 field caught mosquitoes, and both tests concordantly detected P. falciparum in an Anopheles punctulatus mosquito collected from Kaboibus. Both tests thus suggested an overall sporozoite rate of 0.9% (1/107) in the study areas. Subsequently, PCR-RFLP efficiently discriminated P. falciparum from P. vivax for all of the Cytb-PCR positive samples. CONCLUSION: A single step PCR based method has been introduced here that is highly sensitive, efficient and reliable for identifying P. vivax and P. falciparum from mosquitoes. The reliability of the technique was confirmed by its ability to detect Plasmodium as efficiently as those of CS-ELISA and nested PCR. Application of the assay offers the opportunity to detect vector species of Papua New Guinea and may contribute for designing further vector control programmes.

Docosahexaenoic acid ethyl ester enhances 6-hydroxydopamine-induced neuronal damage by induction of lipid peroxidation in mouse striatum.

Superoxide and hydroxyl radicals are implicated in the pathogenesis of Parkinson disease, and induction of lipid peroxidation is an important factor in progression of this disease. Docosahexaenoic acid (DHA) is a key component of the cell membrane, and its peroxidation is inducible due to the double-bond chemical structure. However, DHA has neuroprotective effects. In this study, we examined the effects of intraperitoneal injection (ipi) of DHA ethyl ester (DHA-Et) on 6-hydroxydopamine (6-OHDA)-induced dopamine (DA) reduction in the mouse striatum. DHA-Et ipi for 7 days before and 7 days after a single intracerebroventricular injection of 6-OHDA enhanced 6-OHDA-induced reduction of striatal DA level. On the other hand, ipi of DHA-Et for 7 days increased its concentration in the striatum. Co-injection of DHA-Et and 6-OHDA increased the levels of thiobarbituric acid-reactive substances (a marker of lipid peroxidation) in the striatum. Our results suggest that DHA-Et enhances 6-OHDA-induced DA depression by increasing lipid peroxidation, and that excessive use of DHA-Et may increase the susceptibility of Parkinson disease in animal model.

Thyroglobulin mRNA expression in peripheral blood lymphocytes of healthy subjects and patients with thyroid disease.

BACKGROUND: Thyroglobulin (Tg) mRNA is expressed focally in thyroid tissue. In recent years, the Tg gene has been detected in other tissues, including lymphocytes, although the significance of its presence has not been elucidated yet. We measured Tg mRNA expression in the lymphocytes of healthy subjects and those with thyroid disease. METHODS: Analysis of the quantification of Tg mRNA from 20 healthy subjects and 47 subjects with thyroid disease was carried out by real-time PCR. Furthermore, in cultured lymphocytes we compared changes in Tg mRNA expression following stimulation with TSH. RESULTS: Tg mRNA was detected in the lymphocytes of all subjects. Tg mRNA in the lymphocyte sequence matched that derived from thyroid tissue, and mRNA levels were higher in subjects with thyroid disease than in healthy subjects. Following lymphocyte stimulation, Tg mRNA levels were observed to be increased 2.7-fold in Graves' disease and 1.6-fold in chronic thyroiditis compared to healthy subjects. CONCLUSIONS: Tg mRNA in the lymphocytes was quantified by real-time PCR. The levels of Tg mRNA in the TSH-stimulated lymphocytes were noticeably increased in subjects with thyroid disease. These results suggest an interesting relationship between production of Tg antigen in peripheral blood and autoimmunity in thyroid disease.

Exposure to bisphenol A during embryonic/fetal life and infancy increases oxidative injury and causes underdevelopment of the brain and testis in mice.

We investigated the modifications in endogenous antioxidant capacity and oxidative damage in the brain, liver, kidney and testis in mice exposed to bisphenol A (BPA), an environmental endocrine disrupter. Mice were exposed to BPA throughout embryonic/fetal life and during lactation by feeding their pregnant/lactating mothers BPA at 5 or 10 microg per milliliter of drinking water. At the age of four weeks, male mice were sacrificed. Exposure to BPA increased the activity of catalase and glutathione peroxidase in the liver and kidney, respectively. It also increased thiobarbituric acid-reactive substances in the brain, kidney and testis, and decreased the wet weight of the brain, kidney and testis. Our results suggest that exposure to BPA throughout embryonic/fetal life and during infancy induces tissue oxidative stress and peroxidation, ultimately leading to underdevelopment of the brain, kidney and testis.