A new type of flagellin gene in Pseudomonas putida

Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.     

Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

Background

Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques.

Results

The sequences encoding the variable regions of heavy (V_H) and light (V_L) chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser)₃ and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa). Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab).

Conclusion

This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.     

Characterization of Radioiodinated TISCH: A High-Affinity and Selective Ligand for Mapping CNS D1 Dopamine Receptor

In developing CNS D1 dopamine receptor-imaging agents with improved specificity and longer brain retention, an iodinated D1 ligand was synthesized. In vitro and in vivo radiolabeling studies of a new iodinated benzazepine, TISCH [7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1H-3- benzazepine], an analog of SCH 23390 (7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e), were investigated. After an intravenous injection, the R(+) isomer of TISCH showed high brain uptake in rats (2.20 and 0.57% dose per whole brain at 2 and 60 min, respectively). The striatum/cerebellum ratio increased progressively with time (12 at 60 min). Ex vivo autoradiography of rat brain sections, after intravenous injection of R(+)-[125I]TISCH, displayed the highest uptake in striatum and substantia nigra, regions known to have a high concentration of D1 receptors, whereas the S(-) isomer displayed no specific uptake. Furthermore, the specific uptake can be blocked by pretreatment with SCH 23390. In vitro binding studies using the rat striatum tissue preparation showed high specific and low nonspecific bindings (KD = 0.21 +/- 0.03 nM). The rank order of potency exhibiting high specificity to the D1 receptor was SCH 23390 greater than (+/-)-TISCH greater than (+)-butaclamol = (+/-)-FISCH [7-chloro-8-hydroxy-1-(4'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1 H-3-benzazepine] much greater than WB4101 = spiperone greater than dopamine, serotonin, (+/-)-propranolol, and naloxone. Imaging studies in a monkey with the resolved isomer, R(+)-[123I]TISCH, demonstrated a high uptake in the basal ganglia and prolonged retention. The preliminary data suggest that R(+)-TISCH is selective for the CNS D1 receptor and is potentially useful for in vivo and in vitro pharmacological studies. When labeled with iodine-123, it may be suitable for noninvasive imaging in humans.     

Productivity and Selenium Concentrations in Egg and Tissue of Laying Quails Fed Selenium from Hydroponically Produced Selenium-Enriched Kale Sprout (Brassica oleracea var. alboglabra L.)

This study aimed to determine the effectiveness of Se from hydroponically produced Se-enriched kale sprout (HPSeKS) on productive performance, egg quality, and Se concentrations in egg and tissue of laying quails. Two-hundred quails, 63 days of age, were divided into four groups. Each group consisted of five replicates and each replicate had ten birds, according to a completely randomized design. The experiment lasted for 6 weeks. The dietary treatments were T1 (control diet), T2 (control diet plus 0.2 mg Se/kg from sodium selenite), T3 (control diet plus 0.2 mg Se/kg from Se-enriched yeast), T4 (control diet plus 0.2 mg Se/kg from HPSeKS). The findings revealed that productive performance and egg quality of quails were not altered (p?>?0.05) by Se sources. Whole egg Se concentrations of quails fed Se from HPSeKS and Se-enriched yeast were higher (p?<?0.05) than that of quails fed the control diet. Breast muscle Se concentrations in quails fed Se from HPSeKS were higher (p?<?0.05) than that of quails fed Se from sodium selenite and Se-enriched yeast. Heart tissue Se concentrations of quails fed Se from Se-enriched yeast and HPSeKS were similar (p?>?0.05), but higher (p?<?0.05) than that of quails fed Se from sodium selenite. The results reveal that Se from HPSeKS did not change the performance and egg quality of quails. The effectiveness of Se from HPSeKS was comparable to that of Se-enriched yeast, which was higher than that of Se from sodium selenite.     

Serotypes, antimicrobial susceptibility, and gyr A gene mutation of Campylobacter jejuni isolates from humans and chickens in Thailand

In Thailand, 51% (36/70) Campylobacter jejuni isolates from humans and 68% (47/69) isolates from poultry were classified into 10 Penner serotypes (serotype B, C, R, E, G, A, K, D, I, and L) and 9 serotypes (serotype A, C, I, K, B, E, S, D, and L), respectively. The rate of antimicrobial drug resistance to nalidixic acid, ciprofloxacin, ampicillin, tetracycline, and erythromycin shown by human isolates were 96%, 96%, 29%, 57%, and 14%, while that shown by poultry isolates were 77%, 77%, 22%, 26%, and 17%, respectively. All quinolone-resistant strains contained a mutation in the gyrA gene (T(86)-->I(86)), suggesting that the strains were already widespread in Thailand.     

The Global One Health Paradigm: Challenges and Opportunities for Tackling Infectious Diseases at the Human,Animal, and Environment Interface in Low-Resource Settings

Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years. Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. These interrelated driving forces make it difficult to predict and to prevent zoonotic EIDs. Although significant improvements in environmental and medical surveillance, clinical diagnostic methods, and medical practices have been achieved in the recent years, zoonotic EIDs remain a major global concern, and such threats are expanding, especially in less developed regions. The current Ebola epidemic in West Africa is an extreme stark reminder of the role animal reservoirs play in public health and reinforces the urgent need for globally operationalizing a One Health approach. The complex nature of zoonotic diseases and the limited resources in developing countries are a reminder that the need for implementation of Global One Health in low-resource settings is crucial. The Veterinary Public Health and Biotechnology (VPH-Biotec) Global Consortium launched the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI) in order to address important challenges and needs for capacity building. The inaugural ICOPHAI (Addis Ababa, Ethiopia, 2011) and the second congress (Porto de Galinhas, Brazil, 2013) were unique opportunities to share and discuss issues related to zoonotic infectious diseases worldwide. In addition to strong scientific reports in eight thematic areas that necessitate One Health implementation, the congress identified four key capacity-building needs: (1) development of adequate science-based risk management policies, (2) skilled-personnel capacity building, (3) accredited veterinary and public health diagnostic laboratories with a shared database, and (4) improved use of existing natural resources and implementation. The aim of this review is to highlight advances in key zoonotic disease areas and the One Health capacity needs.     

Hopane triterpenes from the scale insect pathogenic fungus Aschersonia calendulina BCC 23276

Two new hopane-type triterpenenes, 6α,15α,22-trihydroxyhopane (4) and 3β-acetoxy-6α,22-dihydroxyhopane (5), together with the known 6α,22-dihydroxyhopane (1, zeorin), 15α,22-dihydroxyhopane (2, dustanin), and 3β-acetoxy-15α,22-dihydroxyhopane (3) were isolated from the scale insect pathogenic fungus Aschersonia calendulina BCC 23276. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data.     

Particular interaction between pyrimethamine derivatives and quadruple mutant type dihydrofolate reductase of Plasmodium falciparum: CoMFA and quantum chemical calculations studies

Comparative molecular field analysis (CoMFA) was performed on twenty-three pyrimethamine (pyr) derivatives active against quadruple mutant type (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) dihydrofolate reductase of Plasmodium falcipaarum (PfDHFR). The represented CoMFA models were evaluated based on the various three different probe atoms, C(sp3) (+1), O(sp3) (-1) and H (+1), resulting in the best model with combined three types of probe atoms. The statistical results were r(2)(cv) = 0.702, S(press) = 0.608, r(2)(nv) = 0.980, s = 0.156, and r(2)(test-set) = 0.698 which can explain steric contribution of about 50%. In addition, an understanding of particular interaction energy between inhibitor and surrounding residues in the binding pocket was performed by using MP2/6-31G(d,p) quantum chemical calculations. The obtained results clearly demonstrate that Asn108 is the cause of pyr resistance with the highest repulsive interaction energy. Therefore, CoMFA and particular interaction energy analyses can be useful for identifying the structural features of potent pyr derivatives active against quadruple mutant type PfDHFR.     

Halicyclamine A, a marine spongean alkaloid as a lead for anti-tuberculosis agent

In the course of our search for anti-microbial agents against dormant Mycobacterium tuberculosis, halicyclamine A was re-discovered as a lead for anti-tuberculosis agent from a marine sponge of Haliclona sp. on the guidance of the constructed bioassay. Halicyclamine A showed growth inhibition against Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Ra with MICs in the range of 1.0-5.0microg/ml under both aerobic condition and hypoxic condition inducing dormant state. The growth-inhibitory activity of halicyclamine A was bactericidal, and halicyclamine A did not exhibit cross-resistance with the currently used anti-tuberculosis drugs of isoniazid, ethambutol, rifampicin, and streptomycin. Halicyclamine A has been isolated originally as one of the active constituents inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH). Then, in order to elucidate action-mechanism of halicyclamine A, we prepared IMPDH over-expressing strains of M. smegmatis. However, IMPDH was not target for halicyclamine A, because halicyclamine A showed same MIC value against the wild-type M. smegmatis and IMPDH over-expressing strains.     

A Novel Anti-HIV Dextrin–Zidovudine Conjugate Improving the Pharmacokinetics of Zidovudine in Rats

The aim of this study was to investigate a newly synthesized dextrin–zidovudine (AZT) conjugate designed as a sustained release prodrug of AZT for parenteral administration. AZT was first reacted with succinic anhydride to form a succinoylated AZT which was subsequently coupled with dextrin to yield the dextrin–AZT conjugate. The structure of the conjugate was characterized by FT-IR and ¹H-NMR spectroscopy. The drug content of the conjugate was 18.9 wt.%. The release in vitro of free AZT and succinoylated AZT was investigated in buffer solutions at pH 5.5 and 7.4 and in human plasma. AZT and succinoylated AZT release from the conjugate was 1.4% (pH 5.5), 41.7% (pH 7.4) and 78.4% in human plasma after 24 h. Release was complete in human plasma after 48 h. A pharmacokinetic study in rats following intravenous administration of the conjugate showed prolonged plasma levels of AZT compared to free AZT. The use of the conjugate extended the plasma half-life of AZT from 1.3 to 19.3 h and the mean residence time from 0.4 to 23.6 h. Furthermore, the conjugate provided a significant greater area under the plasma concentration-time curve and reduced the systemic clearance of AZT. This study suggested the potential of this novel dextrin–AZT conjugate as a new intravenous preparation of AZT.