A new species of Simulium (Nevermannia) (Diptera, Simuliidae) from Thailand, with keys to members of the Simulium feuerborni species-group in Thailand

Simulium (Nevermannia) maeaiensesp. n. is described on the basis of female, male, pupal and larval specimens collected from Chiang Mai Province, Thailand. This species is assigned to the feuerborni species-group of the subgenus Simulium (Nevermannia), and is distinctive among this species-group in having the female cibarium furnished with numerous dark minute conical processes on the lower part, the female genital fork with a strongly sclerotized horizontal bar on each arm, and six long pupal gill filaments arising nearly at the same level from the common basal stalk and lying in a horizontal plane. Identification keys to seven species of the feuerborni species-group reported from Thailand are provided for females, males, pupae and mature larvae.     

Characterization of Plasmodium knowlesi dihydrofolate reductase-thymidylate synthase and sensitivity to antifolates

Malaria caused by an infection of Plasmodium knowlesi can result in high parasitemia and deaths. Therefore, effective and prompt treatment is necessary to reduce morbidity and mortality. The study aims to characterize P. knowlesi dihydrofolate reductase-thymidylate synthase enzyme (PkDHFR-TS) and its sensitivity to antifolates. The putative Pkdhfr gene was PCR amplified from field isolates collected from the Southern Thailand. Molecular analysis showed 11 polymorphisms in the dhfr domain of the bifunctional dhfr-ts gene. Of these, 1 polymorphism was a non-synonymous substitution (R34L) that had previously been reported but not associated with antifolate resistance. The recombinant PkDHFR-TS enzyme was found to be sensitive to standard antifolates—pyrimethamine and cycloguanil—as well as P218, a registered candidate drug currently first in human clinical trial. Results suggest that antifolates class of compounds should be effective against P. knowlesi infection.     

Production of an antioxidative peptide from hairy basil seed waste by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To circumvent the time-consuming and costly problems associated with natural product extraction, a potential antioxidative peptide selected from hairy basil waste after oil extraction was produced by recombinant DNA technology.

Results

Because the target peptide is short, the recombinant peptide containing seven repeats of the target sequence, QTFQYSRGWTN, and the DNA fragment coding this sequence was cloned into the pQE-30 Xa expression vector and transformed into Escherichia coli. After 6 h of recombinant peptide expression in E. coli, the target peptide was purified by Ni²⁺ affinity chromatography and gel extraction. The expected 15 kDa recombinant target peptide construct was verified by modified dot blot analysis. Compared with the chemically synthesized peptide, the recombinant peptide revealed significantly higher antioxidant activities (p < 0.05), as determined by DPPH and ABTS radical scavenging assays, and in vitro DNA damage induced by hydroxyl radicals.

Conclusion

This approach provides an alternative to produce an antioxidative peptide that provides a potential scaffold for the further development of antioxidative peptides for industrial applications.

     

Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library

Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of V_κ and V_H genes with newly designed primer sets. Initially, we amplified V_κ and V_H genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the V_κ and V_H genes from the framework region 1 to the joining region. The V_κ and V_H genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.     

Synthesis and properties of carboxymethylchitosan hydrogels modified with poly(ester-urethane)

Preparation and properties of carboxymethylchitosan (CMC) modified with polyurethane (PU) containing poly(ethylene adipate) (PEA) as a soft segment is described. Urethane prepolymer was first synthesized by the reaction of PEA with an excess of 1,6-hexamethylene diisocyanate (HDI) to terminate its ends with isocyanate functional groups, followed by chain extension reaction using ethylene glycol as a chain extender. Its chemical structure was characterized by ¹H NMR and FTIR, molecular weight by GPC, and thermal behavior by DSC. To prepare PU-modified CMC (CMC-PU), 1–60 wt% of PU were introduced into the CMC solution of THF:H₂O mixture (50:50 v/v) in the presence of 10 wt% of hexamethylene-1,6-di-(aminocarboxysulfonate) (HDA) to increase network density. Formation of the network structure was confirmed by investigating percent crosslinking and water swelling properties of CMC-PU compared to CMC network without PU. When percent of PU increased from 1 to 60 wt%, percent crosslinking of CMC-PU gradually increased up to 82%, whereas equilibrium water content (EWC) dropped and retained at 1000%. SEM showed microphase separation of PU (10–50 μm) thoroughly dispersed in CMC surface and in the bulk. In addition, CMC-PU exhibited a slight enhancement in toughness properties. Cytotoxicity and biocompatibility tests indicated that CMC-PU was non-toxic.     

Dendritic cells are dysfunctional in patients with operable breast cancer

Background: Dendritic cells (DCs) play a crucial role in presenting antigens to T lymphocytes and inducing cytotoxic T cells. DCs have been studied in patients with breast cancer to define the factors leading to failure of an effective systemic and locoregional anticancer host response. Methods: Purified DCs were obtained from peripheral blood (PB) and lymph nodes (LNs) of women with operable breast cancer, using immunomagnetic bead selection. The stimulatory capacity of DCs in the allogeneic mixed leukocyte reaction (MLR) and autologous T cell proliferation test (purified protein derivative (PPD) as stimulator), the expression of surface markers on DCs and the production of cytokines in vitro by DCs from patients with operable breast cancer and from healthy donors (controls) were studied. Results: 70–75% purified DCs were isolated from PB and LNs. PBDCs and LNDCs from patients with operable breast cancer demonstrated a reduced capacity to stimulate in an MLR, compared with PBDCs from normal donors (p<0.01). Autologous T cell proliferation in patients had a decreased ability to respond to PPD, when compared with controls (p<0.01). However, T cells from patients responded as well as control T lymphocytes in the presence of control DCs. PBDCs and LNDCs from patients expressed low levels of HLA-DR and CD86, and induced decreased interleukin-12 (IL-12) secretion in vitro, compared with DCs from normal donors (p<0.01). Conclusion: These data suggest a defective DC function in patients with operable breast cancer. Switched-off DCs in patients with early breast cancer and decreased IL-12 production may be important factors for progressive tumour growth.     

LINE-1 methylation status of endogenous DNA double-strand breaks

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.     

Molecular properties of membrane-bound FAD-containing D-sorbitol dehydrogenase from thermotolerant Gluconobacter frateurii isolated from Thailand

There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQ-SLDH, having pyrroloquinoline quinone (PQQ), and FAD-SLDH, containing FAD and heme c as the prosthetic groups. FAD-SLDH was purified and characterized from the PQQ-SLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQ-SLDH. The oxidation product of FAD-SLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FAD-SLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FAD-SLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.     

Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.