Non-labeled detection of waterborne pathogen Cryptosporidium parvum using a polydiacetylene-based fluorescence chip

A non-labeling fluorescence sensor system was developed using polydiacetylene (PDA) liposomes composed of 10,12-pentacosadiynoic acid (PCDA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at a 8:2 molar ratio. The PDA liposomes were immobilized onto an amine-coated glass surface using peptide bonding between the carboxyl group of the liposome and the amine group of the glass surface. The optimum ratio of the cross linker (NHS/EDC) to PDA liposome was determined to be 50% for strong immobilization of the liposomes. Residual carboxyl groups of the PDA liposomes were selectively biotinylated, followed by sequential binding of streptavidin and biotin-antibody (bioreceptor). Finally, the performance of the PDA liposome chip was tested for detecting Cryptosporidium parvum, and yielded a detection limit of 1 x 10(3) oocysts/mL. From these results, it is expected that the PDA liposome chip will have high application potential for the detection of waterborne pathogens including C. parvum.     

Increased expression of the F(1)F(o) ATP synthase in response to iron in heart mitochondria

The objective of the present study was to identify mitochondrial components associated with the damage caused by iron to the rat heart. Decreased cell viability was assessed by increased presence of lactate dehydrogenase (LDH) in serum. To assess the functional integrity of mitochondria, Reactive Oxygen Species (ROS), the Respiratory Control Ratio (RCR), ATP and chelatable iron content were measured in the heart. Chelatable iron increased 15-fold in the mitochondria and ROS increased by 59%. Deterioration of mitochondrial function in the presence of iron was demonstrated by low RCR (46% decrease) and low ATP content (96% decrease). Using two dimensional gel electrophoresis (2DE), we identified alterations in 21 mitochondrial proteins triggered by iron overload. Significantly, expression of the alpha, beta, and d subunits of F(1)F(o) ATP synthase increased along with the loss of ATP. This suggests that the F(1)F(o) ATP synthase participates in iron metabolism.     

Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed 1H–15N multidimensional NMR spectroscopy

We used xenon-perturbed ¹H–¹⁵N multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Three limited regions located on the β3-, β5- and β6-strands of dimeric interface were significantly perturbed by urea in the early stage of KSI unfolding, which could lead to dissociation of the dimer into structured monomers at higher denaturant concentration as the interactions in these regions are weakened. The results indicate that the use of xenon as an indirect probe for multidimensional NMR can be a useful method for the equilibrium unfolding study of protein at residue level.     

Expression and characterization of Ca2+-independent lipase from Bacillus pumilus B26

A lipase-producing Bacillus pumilus strain (B26) was isolated from a soil sample collected in Korea. The cloned gene showed that the lipase B26 composed of a 34-amino-acid signal sequence and a 181-amino-acid mature part corresponding to a molecular mass (M_r) of 19,225. Based on the M_r and the protein sequence, the lipase B26 belongs to the lipase family I.4. The optimum temperature and pH of the purified enzyme were 35 °C and 8.5, respectively. The lipase B26 showed a ‘Ca²⁺-independent thermostability and catalytic activity’. These are novel properties observed for the first time in lipase B26 among all bacterial lipases and correspond with the suggestion that this enzyme had no Ca²⁺-binding motif around the catalytic His156 residue. This enzyme seems to be a true lipase based on the experimental results that it could hydrolyze various long-chain triglycerides (C₁₄–C₁₈) and triolein (C_18:1) and that it showed a typical interfacial activation mechanism toward both tripropionin and p-nitrophenyl butyrate.     

Costimulatory Effect of Fas in Mouse T Lymphocytes

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Design,synthesis, and systematic evaluation of 4-arylpiperazine- and 4-benzylpiperidine napthyl ethers as inhibitors of monoamine neurotransmitters reuptake

Two series of 4-arylpiperazine- and 4-benzylpiperidine naphthyl ethers were designed based on structure-activity relationship (SAR) and docking model of reported monoamine neurotransmitters reuptake inhibitors. The compounds were synthesized in 3-simple steps and their biological activities were evaluated. Several compounds were proven to be potent inhibitors of serotonin and norepinephrine reuptake. Computer docking was performed to study the interaction of the most potent compound 35 with human serotonin transporter. The results of the analyses suggest that 4-arylpiperazine- and 4-benzylpiperidine naphthyl ethers might be promising antidepressants worthy of further studies.     

Assessment of Recovery Medium for Production of hCTLA4Ig after Cryopreservation in Transgenic Rice Cells

A reproducible method for cryopreservation of transgenic rice cells (Oryza sativa L. cv. Dongjin) producing recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) has been established. Here, we assessed recovery media and investigated recombinant protein homogeneity after long-term preservation. For recovery of cryopreserved transgenic rice cells, AA medium was suitable in terms of both morphology and production of hCTLA4Ig. There were no differences in cell growth, sugar consumption, and hCTLA4Ig production between non-cryopreserved and cryopreserved cells for up to 1 month. hCTLA4Ig produced from cryopreserved cells was identical that of hCTLA4Ig from non-cryopreserved cells, as determined by analysis of its molecular weight and isoforms. For long-term preservation, cell viability was stably maintained at 61% for 26 months. In conclusion, these results demonstrate the possibility for reproducible cryocell-banking of transgenic rice cells without changes in the characteristics of cells and target proteins.     

The Efficacy of Neurofeedback in Patients with Major Depressive Disorder: An Open Labeled Prospective Study

The purpose of this study was to evaluate the effect of neurofeedback on depressive symptoms and electrophysiological disturbances in patients with major depressive disorder. We recruited participants suffering from depression to evaluate efficacy of left prefrontal beta with alpha/theta training. An 8-week, prospective, open-label study was undertaken. Twenty participants were recruited. The treatment protocol was twice or three times a week training of beta at F3 with alpha/theta at Pz for 8 weeks. When every visit, patients were received beta training for 30 min, and then alpha/theta training for 30 min. Baseline, 4 and 8 week scores of; the Hamilton rating scale for Depression (HAM-D), the Hamilton rating scale for Anxiety (HAM-A), the Beck Depression Inventory (BDI)-II, the Beck Anxiety Inventory (BAI), Clinical global impression-severity (CGI-S), and pre- and post-treatment resting state EEGs were compared. Interhemispheric alpha power asymmetry (A score) was computed for homologous sites F3–F4. Pre- and post-training clinical assessments revealed significant improvements in HAM–D, HAM-A, BDI, and CGI-S scores. Cumulative response rates by HAM-D were 35.0 and 75.0 % at 4 and 8 weeks, respectively, corresponding cumulative remission rates by HAM-D were 15.0 and 55.0 %, respectively. No significant differences were found between pre- and post-treatment A score. Neurofeedback treatment could improve depressive symptoms significantly. In addition, anxiety symptoms and clinical illness severity decreased significantly after neurofeedback treatment. Despite its several limitations, such as, small sample size and lack of a control group, this study suggested neurofeedback has significant effects in patients with major depressive disorder.