Catalytic properties of β-cyclodextrin glucanotransferase from alkalophilic<Emphasis Type="Italic">Bacillus</Emphasis> sp. BL-12 and intermolecular transglycosylation of stevioside

The catalytic properties of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilicBacillus sp. BL-12 specific for the intermolecular transglycosylation of stevioside were investigated. The molecular mass of purified β-CGTase by ultra-filtration and β-cyclodextrin polymer affinity chromatography was estimated to be 90 kDa, which is high compared to other known bacterial CGTases. The optimal pH and temperature were 9.0 and 50°C, respectively, and thermal stability at 40°C was elevated 10-fold in the presence of 1% maltodextrin. The kinetic parameters of the new β-CGTase from alkalophilicBacillus sp. BL-12 indicate that it is more suitable for transglycosylation than the cyclization reaction. Maltodextrin was the most suitable glycosyl donor for transglycosylation of stevioside. The transglycosylation of stevioside was carried out using 60 units of CGTase per gram of maltodextrin, 20 g/L stevioside as the glycosyl acceptor, and 50 g/L maltodextrin as the gycosyl donor at 40°C for 6 h, and a conversion yield of stevioside as high as 76% was obtained.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

Novel adjuvant for immunization against tuberculosis: DNA vaccine expressing <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigen 85A and interleukin-15 fusion product elicits strong immune responses in mice

Objectives

To investigate the potential of interleukin (IL)-15 as a novel adjuvant for Mycobacterium tuberculosis (Mtb) antigen 85A (Ag85A) vaccine.

Results

C57BL/6 mice were intramuscularly immunized three times with a plasmid expressing the Ag85A-IL-15 fusion protein (pcDNA3.1-Ag85A-IL-15), with the empty pcDNA3.1 vector and the pcDNA3.1-Ag85A as control. Mice vaccinated with pcDNA3.1-Ag85A-IL-15 generated more secretory IgA (sIgA) into their lung (209 ± 21 μg/ml) and acquired an enhanced serum IgG response to Ag85A. IgG2a/IgG1 ratios were upregulated, natural killer cell activity was augmented and Ag85A-specific splenic T cell proliferation was enhanced in these mice as well. Vaccination with pcDNA3.1-Ag85A-IL-15 promoted the polarization of CD4⁺ T cells towards a Th1 type in the spleen, and significantly upregulated the serum level of interferon (IFN)-γ (458 ± 98 pg/ml), a typical Th1 cytokine. IFN-γ-expressing CD8⁺ cells were also increased in the spleen after pcDNA3.1-Ag85A-IL-15 immunization.

Conclusions

A superior immune type I response in mice vaccinated with plasmid Ag85A-IL-15 has been achieved.

     

Inversion polymorphism and a new polytene chromosome map of <Emphasis Type="Italic">Zaprionus indianus</Emphasis> Gupta (1970) (Diptera: drosophilidae)

Zaprionus indianus is a recent invader in Brazil and was probably introduced from the West Afrotropical zone. So far, studies regarding its chromosomal polymorphism were limited to India. We found that Brazilian populations were very different from Indian ones. Five new inversions have been discovered. In(II)A, already described in India, where it is quite common, has also been found in Brazil, where it is very rare. The X-chromosome has three inversions; In(X)Na, In(X)Ke and In(X)Eg, which are frequent in all Brazilian populations studied. In every case, we observed strong linkage disequilibrium among these gene arrangements. During the primary collection period (2001–2002), we noticed a significant positive correlation between the frequency of these inversions and latitude, but this was not confirmed in later investigations. Rearrangement In(IV)EF was also common in all populations, while inversion In(V)B was only found in southern populations. Our data suggest that the founders that recently invaded Brazil were polymorphic for the six inversions observed. The place of origin might be identified more precisely by investigating West African populations. In order to facilitate further investigations, we present an updated polytene chromosome photomap, locating the breakpoints of every inversion observed in Brazilian populations. Galina Ananina and Cláudia Rohde contributed equally to this work     

G Protein betagamma subunits augment UVB-induced apoptosis by stimulating the release of soluble heparin-binding epidermal growth factor from human keratinocytes

UV radiation induces various cellular responses by regulating the activity of many UV-responsive enzymes, including MAPKs. The betagamma subunit of the heterotrimeric GTP-binding protein (Gbetagamma) was found to mediate UV-induced p38 activation via epidermal growth factor receptor (EGFR). However, it is not known how Gbetagamma mediates the UVB-induced activation of EGFR, and thus we undertook this study to elucidate the mechanism. Treatment of HaCaT-immortalized human keratinocytes with conditioned medium obtained from UVB-irradiated cells induced the phosphorylations of EGFR, p38, and ERK but not that of JNK. Blockade of heparin-binding EGF-like growth factor (HB-EGF) by neutralizing antibody or CRM197 toxin inhibited the UVB-induced activations of EGFR, p38, and ERK in normal human epidermal keratinocytes and in HaCaT cells. Treatment with HB-EGF also activated EGFR, p38, and ERK. UVB radiation stimulated the processing of pro-HB-EGF and increased the secretion of soluble HB-EGF in medium, which was quantified by immunoblotting and protein staining. In addition, treatment with CRM179 toxin blocked UV-induced apoptosis, but HB-EGF augmented this apoptosis. Moreover, UVB-induced apoptosis was reduced by inhibiting EGFR or p38. The overexpression of Gbeta(1)gamma(2) increased EGFR-activating activity and soluble HB-EGF content in conditioned medium, but the sequestration of Gbetagamma by the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2ct) produced the opposite effect. The activation of Src increased UVB-induced, Gbetagamma-mediated HB-EGF secretion, but the inhibition of Src blocked that. Overexpression of Gbetagamma increased UVB-induced apoptosis, and the overexpression of GRK2ct decreased this apoptosis. We conclude that Gbetagamma mediates UVB-induced human keratinocyte apoptosis by augmenting the ectodomain shedding of HB-EGF, which sequentially activates EGFR and p38.     

cDNA cloning of translationally controlled tumor protein/histamine releasing factor (TCTP/HRF) from the intertidal harpacticoid copepod Tigriopus japonicus.

We synthesized a cDNA library from the intertidal copepod Tigriopus japonicus, converted it to phagemids and sequenced expressed sequence tags (ESTs). Of these, Tigriopus translationally controlled tumor protein/histamine releasing factor (TCTP/HRF) was further characterized. The Tigriopus TCTP/HRF gene encoded 172 amino acid residues and showed high similarity to Drosophila but moderate similarity to other annelids (e.g. Brugia, Wuchereria and C. elegans). The Tigriopus TCTP/HRF gene appeared in the same clade as the annelids. Here, we describe the analysis of the Tigriopus TCTP/HRF gene.     

Caspofungin reduces the incidence of fungal contamination in cell culture

Fungal contamination is a major problem in cell culture, and the antifungal compounds currently in use can affect cultured cells. Echinocandins are antifungal drugs that inhibit fungal cell wall synthesis by targeting an enzyme that has no counterpart in mammalian cells. We evaluated whether the echinocandin caspofungin affected the growth or morphology of six murine cell lines (a macrophage-like cell line (J774.16) and five hybridoma lines), or primary human endothelial cells. The antifungal did not influence cellular characteristics at concentrations less than 512 μg/ml, while effectively reducing the incidence of fungal contamination. Also, caspofungin did not affect the production of antibody by hybridoma cells, or alter the cytokine production of J774.16 cells, although modest increases in IL-4 and IFN-γ occurred upon LPS stimulation. Hence, echinocandins appear to be relatively non-toxic, and protect against fungal contamination in cell culture.     

What governs the reaction center excitation wavelength of photosystems I and II?

The sun’s spectrum harvested through photosynthesis is the primary source of energy for life on earth. Plants, green algae, and cyanobacteria—the major primary producers on earth—utilize reaction centers that operate at wavelengths of 680 and 700 nm. Why were these wavelengths “chosen” in evolution? This study analyzes the efficiency of light conversion into chemical energy as a function of hypothetical reaction center absorption wavelengths given the sun’s spectrum and the overpotential cost associated with charge separation. Surprisingly, it is found here that when taking into account the empirical charge separation cost the range 680–720 nm maximizes the conversion efficiency. This suggests the possibility that the wavelengths of photosystem I and II were optimized at some point in their evolution for the maximal utilization of the sun’s spectrum.     

Characterization and development of EST-SSR markers to study the genetic diversity and populations analysis of Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.)

In recent years, Jerusalem artichoke has received widespread attention as a novel source of sugar, biofuel, and animal feed. Currently, only few gDNA-SSRs derived from sunflower were verified in the Jerusalem artichoke; therefore, it is particularly important to develop SSR primer markers that belonged to Jerusalem artichoke resources. Using EST data to develop EST-SSR markers is simple and effective. In order to understand the general characteristics of SSR markers in Jerusalem artichoke EST sequences and accelerate the use of SSR markers in Jerusalem artichoke research. This study used 40,370 sequenced unigene fragments and MISA software to identify SSR loci. The 48 pairs of EST-SSR primers assessed for the identification of 45 varieties of Jerusalem artichoke. Cluster, genetic diversity parameters and AMOVA analysis was conducted using the genetic similarity coefficient, revealing genetic differences between 48 genetic material. A total of 1204 SSR loci were identified with 13 different types of repeats, distributed among 1020 EST sequences, of which trinucleotide repeats were the most common, accounting for 38.21% of the total SSR loci. Among the 44 repeat motifs, AG/CT, AAG/CTT, and ATC/ATG motifs had the highest frequencies, accounting for 22.45, 14.71, and 7.84% of all motifs, respectively. From these sequences, 48 pairs of EST-SSR primers were designed, and 22 primer pairs for loci with high polymorphism were selected to analyze the genetic diversity of 45 Jerusalem artichoke germplasm sources. The results indicated that the variation range of the effective number of alleles for 22 primers ranged between 1.7502 and 4.5660. The Shannon’s information index ranged between 0.6200 and 1.6423. The variation range of PIC ranged between 0.3121 and 0.6662 with an average of 0.5184. Cluster analysis was conducted using the genetic similarity coefficient, revealing significant genetic differences between Asian and European genetic material. Cluster analysis revealed a relationship between the genotypes and geographic origins of the Jerusalem artichoke. The results of AMOVA as well as the genetic identity and genetic distance in the Jerusalem artichoke population showed that there presented certain genetic heterogeneity in Jerusalem artichoke genetic structure of 45 samples from seven different geographic populations. The Jerusalem artichoke EST-SSR marker system established in this study provides an effective molecular marker system for future research focused on Jerusalem artichoke genetic diversity and the breeding of new varieties.