Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach

Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.     

Cyclic ADP ribose-dependent Ca2+ release by group I metabotropic glutamate receptors in acutely dissociated rat hippocampal neurons

Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5) exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca²⁺. In this study, we characterized the cellular mechanisms underlying Ca²⁺ mobilization induced by (RS)-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist) in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca²⁺ from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR), while the PLC/IP₃ signaling pathway was not involved in Ca²⁺ mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4), led to transient Ca²⁺ mobilization by mGluR5 and Ca²⁺ influx through L-type Ca²⁺ channels. We found no evidence that mGluR5-mediated Ca²⁺ release and Ca²⁺ influx through L-type Ca²⁺ channels interact to generate supralinear Ca²⁺ transients. Our study provides novel insights into the mechanisms of intracellular Ca²⁺ mobilization by mGluR5 in the somata of hippocampal neurons.     

Cryopreservation of amniotic fluid-derived stem cells using natural cryoprotectants and low concentrations of dimethylsulfoxide

Amniotic fluid-derived stem cells (AFSCs) are a potential cell source for therapeutic applications. They can be easily mass produced, cryopreserved and shipped to clinics for immediate use. However, one major obstacle to the manufacturing of clinical grade stem cells is the need for current good manufacturing practices for cryopreservation, storage, and distribution of these cells. Most current cryopreservation methods used for stem cells include the potentially toxic cryoprotectant (CPA) dimethylsulfoxide (Me₂SO) in the presence of animal serum proteins that prevent direct use of these cells in human therapeutic applications. To avoid any potential cryoprotectant related complications, it will be essential to develop non-toxic CPAs or reduce CPA concentration in the freezing media used. In this study, we assessed the use of disaccharides, antioxidants and caspase inhibitors for cryopreservation of AFSCs in combination with a reduced concentration of Me₂SO. The thawed cells were tested for viability with MTT assays and a growth curve was created to measure population doubling time. In addition, we performed flow cytometry analysis for cell surface antigens, RT-PCR for mRNA expression of stem cell markers, and assays to determine the myogenic differentiation potential of the cells. A statistically significant (p < 0.05) increase in post-thawed cell viability in solutions containing trehalose, catalase and _ZVAD-fmk with 5% Me₂SO was observed. The solutions containing trehalose and catalase with 5% or 2.5% (v/v) Me₂SO produced results similar to those for the control (10% (v/v) Me₂SO and 30% FBS) in terms of culture growth, expression of cell surface antigens and mRNA expression of stem cell markers in AFSCs cryopreserved for a minimum of 3 weeks. Thus, AFSCs can be cryopreserved with 1/4 the standard Me₂SO concentration with the addition of disaccharides, antioxidants and caspase inhibitors. The use of Me₂SO at low concentrations in cell freezing solutions may support the development of clinical trials of AFSCs.     

Magnesium Supplementation through Seaweed Calcium Extract Rather than Synthetic Magnesium Oxide Improves Femur Bone Mineral Density and Strength in Ovariectomized Rats

Commercially available seaweed calcium extract can supply high amounts of calcium as well as significant amounts of magnesium and other microminerals. The purpose of this study was to investigate the degree to which the high levels of magnesium in seaweed calcium extract affects the calcium balance and the bone status in ovariectomized rats in comparison to rats supplemented with calcium carbonate and magnesium oxide. A total of 40 Sprague-Dawley female rats (7?weeks) were divided into four groups and bred for 12?weeks: sham-operated group (Sham), ovariectomized group (OVX), ovariectomized with inorganic calcium and magnesium supplementation group (OVX-Mg), and ovariectomized with seaweed calcium and magnesium supplementation group (OVX-SCa). All experimental diets contained 0.5% calcium. The magnesium content in the experimental diet was 0.05% of the diet in the Sham and OVX groups and 0.1% of the diet in the OVX-Mg and OVX-SCa groups. In the calcium balance study, the OVX-Mg and OVX-SCa groups were not significantly different in calcium absorption compared to the OVX group. However, the femoral bone mineral density and strength of the OVX-SCa group were higher than those of the OVX-Mg and OVX groups. Seaweed calcium with magnesium supplementation or magnesium supplementation alone did not affect the serum ALP and CTx levels in ovariectomized rats. In summary, consumption of seaweed calcium extract or inorganic calcium carbonate with magnesium oxide demonstrated the same degree of intestinal calcium absorption, but only the consumption of seaweed calcium extract resulted in increased femoral bone mineral density and strength in ovariectomized rats. Our results suggest that seaweed calcium extract is an effective calcium and magnesium source for improving bone health compared to synthetic calcium and magnesium supplementation.     

Prevalence of Clonorchis sinensis metacercariae in freshwater fish from three latitudinal regions of the Korean Peninsula

A large-scale survey was conducted to investigate the infection status of fresh water fishes with Clonorchis sinensis metacercariae (CsMc) in 3 wide regions, which were tentatively divided by latitudinal levels of the Korean peninsula. A total of 4,071 freshwater fishes were collected from 3 regions, i.e., northern (Gangwon-do: 1,543 fish), middle (Chungcheongbuk-do and Gyeongsangbuk-do: 1,167 fish), and southern areas (Jeollanam-do, Ulsan-si, and Gyeongsangnam-do: 1,361 fish). Each fish was examined by the artificial digestion method from 2003 to 2010. In northern areas, only 11 (0.7%) fish of 2 species, Pungtungia herzi and Squalidus japonicus coreanus from Hantan-gang, Cheolwon-gun, Gangwon-do were infected with av. 2.6 CsMc. In middle areas, 149 (12.8%) fish were infected with av. 164 CsMc. In southern areas, 538 (39.5%) fish were infected with av. 159 CsMc. In the analysis of endemicity in 3 regions with an index fish, P. herzi, 9 (6.2%) of 146 P. herzi from northern areas were infected with av. 2.8 CsMc. In middle areas, 34 (31.8%) of 107 P. herzi were infected with av. 215 CsMc, and in southern areas, 158 (92.9%) of 170 P. herzi were infected with av. 409 CsMc. From these results, it has been confirmed that the infection status of fish with CsMc is obviously different among the 3 latitudinal regions of the Korean peninsula with higher prevalence and burden in southern regions.     

Echinostoma ilocanum infection in Oddar Meanchey Province, Cambodia

Fecal examinations using the Kato Katz technique were performed on a total of 1,287 villagers (945 students and 342 general inhabitants) of Oddar Meanchey Province, Cambodia in May 2007 and November 2009. The overall intestinal helminth egg positive rate was 23.9%, and the most prevalent helminth species was hookworms (21.6%). Other helminth eggs detected included echinostomes (1.0%), Enterobius vermicularis (0.8%), small trematode eggs (0.7%), which may include Opisthorchis viverrini and Haplorchis spp., and Hymenolepis nana (0.4%). In order to recover adult echinostomes, we treated 2 patients with 10-15 mg/kg praziquantel and purged. Total 14 adult echinostomes, 1 and 13 worms from each patient, were collected. The echinostomes characteristically had 49-51 collar spines and 2 round or slightly lobated testes. They were identified as Echinostoma ilocanum (Garrison, 1908) Odhner, 1911. So far as literature are concerned, this is the first record on the discovery of human E. ilocanum infection in Cambodia.     

Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.     

Molecular identification of Taenia tapeworms by Cox1 gene in Koh Kong, Cambodia

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.     

Therapeutic effects of metformin in breast cancer: involvement of the immune system?

Breast cancer and associated diabetes mellitus have gained raising interest as an elevated risk of breast cancer prognosis resulting in increased mortality in diabetic patients. In this context, the long-acting insulin analog glargine and other antidiabetics have been discussed to promote tumorigenesis. In contrast, the biguanide class oral antidiabetic metformin has been shown capable of enhancing cell cycle arrest and inducing apoptosis as well as reducing growth factor signaling. Consequently, several studies are underway to evaluate a possible role of metformin in breast cancer treatment. Although mechanisms involved are not definitely clear yet, here, we discuss metformin’s anticancer effects including the potential impact of the immune system.