Coenzyme Q10 inhibits mitochondrial complex-1 down-regulation and nuclear factor-kappa B activation

We have used control-homozygous weaver mutant, and -heterozygous weaver mutant mice in order to explore the basic molecular mechanism of neurodegeneration and the neuroprotective potential of coenzyme Q(10). Homozygous weaver mutant mice exhibited progressive neurodegeneration in the hippocampus, striatum, and cerebellum, and a reduction in the striatal levels of dopamine and coenzyme Qs (Q(9) and Q(10)) without any significant changes in norepinephrine and serotonin. Mitochondrial complex-1 was down regulated; whereas nuclear factor-kappa B was up regulated in homozygous weaver mutant mice. Rotenone inhibited complex-1, enhanced nuclear factor-kappa B, and caused apoptosis in human dopaminergic (SK-N-SH) neurons; whereas nuclear factor-kappa B antibody suppressed rotenone-induced apoptosis, suggesting that enhancing coenzyme Q(10) synthesis and suppressing the induction of NF-kappa B, may provide neuroprotection.     

Platelet serotonin transporter in schizophrenic patients with and without neuroleptic treatment

Among various hypotheses put forth to account for the etiology of schizophrenia, the abnormal function of serotonergic system has recently gained marked interest. Our previous study showed that drug-free schizophrenic patients had a significant increase in maximum numbers (B(max)) of platelet 5-HT(2A) receptors that declined to normal level after treatment with different neuroleptic drugs. To elucidate the role of the serotonin system in schizophrenia, the serotonin transporters on human platelets were examined in this study. Platelet serotonin transporters obtained from normal control subjects and schizophrenic patients were identified by using [(3)H]imipramine as the radioligand and fluoxetine to define the non-specific binding. The data showed that the mean B(max) of serotonin transporter sites for schizophrenic patients without neuroleptic therapy was significantly higher than in normal controls. The B(max) values for schizophrenic patients on phenothiazine, butyrophenone, thioxanthene and serotonin-dopamine antagonist (SDA) therapies were significantly lower than the B(max) values obtained from schizophrenic patients without neuroleptic therapy, and were comparable to those found in normal control subjects. The dissociation equilibrium constant (K(d)) values in all subject groups remained unchanged. The effect of various medication periods on platelet serotonin transporters was also studied. We found that, B(max) values of 1-4 weeks, 1-4 months, 4-12 months and >1 year of neuroleptic therapies were significantly decreased when compared with the unmedicated group. Significant reduction of brief psychiatric rating scale (BPRS) occurred in all types of neuroleptics and every period of drug treatments compared with the unmedicated group. The present results indicate that alteration of platelet serotonin transporters is associated with schizophrenia. Treatment with various types of neuroleptics suppresses the hypersensitivity of platelet serotonin transporters. The mechanisms of how neuroleptics achieve their therapeutic effects, whether they act via or modulate serotonin system in certain brain area, still need to be further evaluated.     

Evaluation of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the identification of Campylobacter strains isolated from poultry in Thailand

We have recently developed a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for identifying Campylobacter jejuni, C. coli and C. fetus. In the present study, the applicability of this assay was evaluated with 34 Campylobacter-like organisms isolated from poultry in Thailand for species identification and was compared with other assays including API Campy, 16S rRNA gene sequence, and hippuricase (hipO) gene detection. Of the 34 strains analyzed, 20, 10 and 1 were identified as C. jejuni, C. coli, and Arcobacter cryaerophilus, respectively, and 3 could not be identified by API Campy. However, 16S rRNA gene analysis, showed that all 34 strains are C. jejuni/coli. To discriminate between these 2 species, the hipO gene, which is specifically present in C. jejuni, was examined by PCR and was detected in 20 strains, which were identified as C. jejuni by API Campy but not in the remaining 14 strains. Collective results indicated that 20 strains were C. jejuni whereas the 14 strains were C. coli. When the cdt gene-based multiplex PCR was employed, however, 19, 20 and 19 strains were identified as C. jejuni while 13, 14 and 13 were identified as C. coli by the cdtA, cdtB and cdtC gene-based multiplex PCR, respectively. Pulsed-field gel electrophoresis revealed that C. jejuni and C. coli strains analyzed are genetically diverse. Taken together, these data suggest that the cdt gene-based multiplex PCR, particularly cdtB gene-based multiplex PCR, is a simple, rapid and reliable method for identifying the species of Campylobacter strains.     

Regulation of cullin RING E3 ubiquitin ligases by CAND1 in vivo

Cullin RING ligases are multi-subunit complexes consisting of a cullin protein which forms a scaffold onto which the RING protein Rbx1/2 and substrate receptor subunits assemble. CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo. Two models have been proposed to explain this requirement: (i) CAND1 sequesters cullin proteins and thus prevents autoubiquitination of substrate receptors, and (ii) CAND1 is required to promote the exchange of bound substrate receptors. Using mammalian cells, we show that CAND1 is predominantly cytoplasmically localized and that cullins are the major CAND1 interacting proteins. However, only small amounts of CAND1 bind to Cul1 in cells, despite low basal levels of Cul1 neddylation and approximately equal cytoplasmic endogenous protein concentrations of CAND1 and Cul1. Compared to F-box protein substrate receptors, binding of CAND1 to Cul1 in vivo is weak. Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding. In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.     

Lysophosphatidic acid enhances interleukin-13 gene expression and promoter activity in T cells

Airway smooth muscle cells (ASMC) are a source of inflammatory chemokines that may propagate airway inflammatory responses. We investigated the production of the CXC chemokine growth-related oncogene protein-alpha (GRO-alpha) from ASMC induced by cytokines and the role of MAPK and NF-kappaB pathways. ASMC were cultured from human airways, grown to confluence, and exposed to cytokines IL-1beta and TNF-alpha after growth arrest. GRO-alpha release, measured by ELISA, was increased by >50-fold after IL-1beta (0.1 ng/ml) or 5-fold after TNF-alpha (1 ng/ml) in a dose- and time-dependent manner. GRO-alpha release was not affected by the T helper type 2 cytokines IL-4, IL-10, and IL-13. IL-1beta and TNF-alpha also induced GRO-alpha mRNA expression. Supernatants from IL-1beta-stimulated ASMC were chemotactic for neutrophils; this effect was inhibited by anti-GRO-alpha blocking antibody. AS-602868, an inhibitor of IKK-2, and PD-98059, an inhibitor of ERK, inhibited GRO-alpha release and mRNA expression, whereas SP-600125, an inhibitor of JNK, reduced GRO-alpha release without effect on mRNA expression. SB-203580, an inhibitor of p38 MAPK, had no effect. AS-602868 but not PD-98059 or SP-600125 inhibited p65 DNA-binding induced by IL-1beta and TNF-alpha. By chromatin immunoprecipitation assay, IL-1beta and TNF-alpha enhanced p65 binding to the GRO-alpha promoter, which was inhibited by AS-602868. IL-1beta- and TNF-alpha-stimulated expression of GRO-alpha from ASMC is regulated by independent pathways involving NF-kappaB activation and ERK and JNK pathways. GRO-alpha released from ASMC participates in neutrophil chemotaxis.     

A VHH That Neutralizes the Zinc Metalloproteinase Activity of Botulinum Neurotoxin Type A

The current treatment of botulism is to administer animal-derived antitoxin, which frequently causes severe adverse reactions in the recipients. In this study, a heavy chain antibody fragment (VH/V^HH) phage display library was constructed by amplification of the immunoglobulin genes of a nonimmune camel, Camelus dromedarius, using primers specific to human VH gene segments. A recombinant light chain of type A botulinum toxin, BoTxA/LC, with zinc endoprotease activity was used in phage bio-panning to select phage clones displaying BoTxA/LC-bound VH/V^HH. Soluble VH/V^HH were produced and purified from 10 VH/V^HH phagemid-transformed E. coli clones. Complementary determining regions (CDRs) and immunoglobulin frameworks (FRs) of the 10 camel VH/V^HH-deduced amino acid sequences were determined. FR2 sequences of two clones showed a hallmark of camel V^HH, i.e. (F/Y)⁴²E⁴⁹R⁵⁰(G/F)⁵². The remaining eight clones had an FR2 amino acid tetrad of conventional VH, i.e. V⁴²G⁴⁹L⁵⁰W⁵². V^HH of one clone (V^HH17) neutralized the SNAP25 hydrolytic activity of BoTxA/LC, whereas mouse polyclonal anti-BoTxA/LC did not have such activity. Mimotope sequences of V^HH17 matched with the 194–206 amino acid residues of BoTxA/LC, which are located near the S′1 subsite of the catalytic cleft of the enzyme. Molecular docking revealed that CDR3 of the V^HH17 bound to epitope in the toxin enzymatic cleft. Therefore, the BoTxA/LC neutralization by the V^HH17 should be due to the V^HH insertion into the enzymatic cleft of the toxin, which is usually inaccessible to a conventional antibody molecule. This antibody fragment warrants further development as a therapeutic agent for botulism.     

Characterization of the Cullin7 E3 ubiquitin ligase--heterodimerization of cullin substrate receptors as a novel mechanism to regulate cullin E3 ligase activity

Cul1 and Cul7 are cullin E3 ubiquitin ligase scaffold proteins. Cul1 is known to form a complex with the RING domain protein Rbx1 and one of approximately 70 different F-box proteins. F-box proteins function as substrate receptor subunits and recruit numerous substrates for poly-ubiquitination. Similarly to Cul1, Cul7 interacts with Rbx1, however, only one F-box protein, Fbxw8, has been shown to bind to Cul7. To date only few Cul7 E3 ubiquitin ligase substrates, including cyclin D1, IRS-1 and GRASP65, have been reported, and using Fbxw8 affinity purification, we were unable to identify additional substrate proteins. Here we provide evidence for a model in which Cul7-Rbx1 can promote the ubiquitination of Cul1 substrates by forming high order complexes with Cul1-Rbx1. Binding of Cul1-Rbx1 to Cul7-Rbx1 is mediated via heterodimerization of Fbxw8 with other F-box proteins which function to recruit substrates into the E3 ligase complex. The formation of this high order complex is likely to increase polyubiquitination efficiency.     

Mouse Model of Cat Allergic Rhinitis and Intranasal Liposome-Adjuvanted Refined Fel d 1 Vaccine

Cats (Felis domesticus) are rich source of airborne allergens that prevailed in the environment and sensitized a number of people to allergy. In this study, a mouse model of allergic rhinitis caused by the cat allergens was developed for the first time and the model was used for testing therapeutic efficacy of a novel intranasal liposome-entrapped vaccines made of native Fel d 1 (major cat allergen) in comparison with the vaccine made of crude cat hair extract (cCE). BALB/c mice were sensitized with cCE mixed with alum intraperitoneally and intranasally. The allergic mice were treated with eight doses of either liposome (L)-entrapped native Fel d 1 (L-nFD1), L-cCE), or placebo on every alternate day. Vaccine efficacy evaluation was performed one day after provoking the treated mice with aerosolic cCE. All allergenized mice developed histological features of allergic rhinitis with rises of serum specific-IgE and Th2 cytokine gene expression. Serum IgE and intranasal mucus production of allergic mice reduced significantly after vaccination in comparison with the placebo mice. The vaccines also caused a shift of the Th2 response (reduction of Th2 cytokine expressions) towards the non-pathogenic responses: Th1 (down-regulation of the Th1 suppressive cytokine gene, IL-35) and Treg (up-regulation of IL-10 and TGF-β). In conclusions, a mouse model of allergic rhinitis to cat allergens was successfully developed. The intranasal, liposome-adjuvanted vaccines, especially the refined single allergen formulation, assuaged the allergic manifestations in the modeled mice. The prototype vaccine is worthwhile testing further for clinical use in the pet allergic patients.     

Diversity of root-associated arbuscular mycorrhizal fungal communities in a rubber tree plantation chronosequence in Northeast Thailand

Rubber tree (Hevea brasiliensis) is of major economic importance in Southeast Asia and for small land holders in Thailand in particular. Due to the high value of latex, plantations are expanding into unsuitable areas, such as the northeast province of Thailand where soil fertility is very low and therefore appropriate management practices are of primary importance. Arbuscular mycorrhizal fungi (AMF) contribute to plant growth through a range of mechanisms and could play a key role in a more sustainable management of the rubber plantations. We described the diversity of AMF associated with rubber tree roots in Northeast Thailand in relation to tree age and soil parameters along a chronosequence of rubber tree plantations. Cassava fields were included for comparison. Rubber tree and cassava roots harbored high diversity of AMF (111 Virtual Taxa, VT), including 20 novel VT. AMF VT richness per sample was consistently high (per site mean 16 to 21 VT per sample) along the chronosequence and was not related to soil properties. The composition of AMF communities differed between cassava and rubber tree plantations and was influenced by soil texture and nutrient content (sand, K, P, Ca). AMF community composition gradually shifted with the age of the trees. Our results suggest that the high diversity of AMF in this region is potentially significant for maintaining high functionality of AMF communities.     

Generation of human single-chain variable fragment antibodies specific to dengue virus non-structural protein 1 that interfere with the virus infectious cycle

Severe forms of dengue virus (DENV) infection frequently cause high case fatality rate. Currently, there is no effective vaccine against the infection. Clinical cases are given only palliative treatment as specific anti-DENV immunotherapy is not available and it is urgently required. In this study, human single-chain variable fragment (HuScFv) antibodies that bound specifically to the conserved non-structural protein-1 (NS1) of DENV and interfered with the virus replication cycle were produced by using phage display technology. Recombinant NS1 (rNS1) of DENV serotype 2 (DENV2) was used as antigen in phage bio-panning to select phage clones that displayed HuScFv from antibody phage display library. HuScFv from two phagemid transformed E. coli clones, i.e., clones 11 and 13, bound to the rNS1 as well as native NS1 in both secreted and intracellular forms. Culture fluids of the HuScFv11/HuScFv13 exposed DENV2 infected cells had significant reduction of the infectious viral particles, implying that the antibody fragments affected the virus morphogenesis or release. HuScFv epitope mapping by phage mimotope searching revealed that HuScFv11 bound to amino acids 1–14 of NS1, while the HuScFv13 bound to conformational epitope at the C-terminal portion of the NS1. Although the functions of the epitopes and the molecular mechanism of the HuScFv11 and HuScFv13 require further investigations, these small antibodies have high potential for development as anti-DENV biomolecules.