Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase

H⁺-translocating pyrophosphatase (H⁺-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PP_i) hydrolysis. Vigna radiata H⁺-PPase (VrH⁺-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H⁺-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H⁺-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH⁺-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K⁺-mediated stimulation of H⁺-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PP_i hydrolysis, proton transport, expression, and K⁺ stimulation of H⁺-PPase.     

Novel recombinant human lactoferrin: Differential activation of oxidative stress related gene expression

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins.     

Transgenic lines of melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) expressing antifungal protein and chitinase genes exhibit enhanced resistance to fungal pathogens

The oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) is an important fruit crop in the tropical and subtropical regions. However, oriental melon production is severely decreased by fungal diseases. In this study, antifungal protein (AFP) and chitinase (CHI) fusion genes were introduced into oriental melons to control fungal diseases caused by Rhizoctonia solani and Fusarium oxysporum. Transformation of oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) with Agrobacterium tumefaciens strain LBA4404 containing antifungal protein (AFP) and chitinase (CHI) fusion genes under the control of the cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene as a selectable marker was performed. Cotyledon explants of oriental melon were inoculated by Agrobacterium suspensions with pBI121–AFP–CHI and cultured in a regeneration medium. After regeneration, genomic DNA polymerase chain reaction (PCR) was conducted to confirm the presence of putative transgenic shoots. Southern blot analysis confirmed that the AFP–CHI fusion gene was incorporated into the genomic DNA of the PCR-positive lines. RT-PCR analysis showed that the AFP–CHI fusion gene was expressed in the individual transgenic lines. Western blot analysis revealed the accumulation of CHI protein in leaves. A segregation analysis of the T₁ generation confirmed the inheritance of the transgene. Our results demonstrated that the AFP–CHI fusion gene was effective in protecting the transgenic melon plants against fungal disease caused by Rhizoctonia solani and Fusarium oxysporum.     

Zinc oxide-doped poly(urethane) spider web nanofibrous scaffold via one-step electrospinning: a novel matrix for tissue engineering

Zinc oxide (ZnO) nanostructures have been commonly studied for electronic purposes due to their unique piezoelectric and catalytic properties; however, recently, they have been also exploited for biomedical applications. The purpose of this study was to fabricate ZnO-doped poly(urethane) (PU) nanocomposite via one-step electrospinning technique. The utilized nanocomposite was prepared by using colloidal gel composed of ZnO and PU, and the obtained mats were vacuum dried at 60 °C overnight. The physicochemical characterization of as-spun composite nanofibers was carried out by X-ray diffraction pattern, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, electron probe microanalysis, and transmission electron microscopy, whereas the thermal behavior was analyzed by thermogravimetric analysis. The viability, attachment, and proliferation of NIH 3T3 mouse fibroblast cells on the ZnO/PU composite nanofibers were analyzed by in vitro cell compatibility test. The morphological features of the cells attached on nanofibers were examined by Bio-SEM. We conclude that the electrospun nanofibrous scaffolds with unique spider nets had good biocompatibility. Cytotoxicity experiments indicated that the mouse fibroblasts could attach to the nanocomposite after being cultured. Thus, the current work demonstrates that the as-synthesized ZnO/PU hybrid nanofibers represent a promising biomaterial to be exploited for various tissue engineering applications.     

Kinetic Properties of a Dipeptidase from Streptococcus cremoris

Some kinetic properties of a dipeptidase purified from a cell-free extract of Streptococcus cremoris H 61 were investigated. The Km values of this enzyme for various dipeptides were divided into 3 groups. Group 1 comprised mainly of neutral dipeptides, such as Leu-Gly, Leu-Leu and Leu-Ala, which had relatively low Km values (in the range 4.0-6.6 mm). Group 2 consisted of dipeptides with aromatic large amino acids either at the N- or C-terminal positions, like Leu-Phe, Phe-Ala and Leu-Tyr, which had very low Km values (in the range 1.0-2.4 mm). Group 3 was made up by dipeptides with acidic or basic amino acids at the N-terminals; His-Ala and Glu-Val were typical of this group. These had very high Km values (in the range 10–20 mm). Substantial substrate competition was found to exist in the presence of His-Ala. Bestatin inhibited the enzyme competitively with Leu-Gly and was found to have an apparent Ki value of 3.0 × 10^?8 m for the enzyme. Further, the enzyme was completely inhibited by EDTA at a concentration of 2.0 × 10^?5 m. On the other hand, once the activity was inhibited by EDTA, it could be restored by Co²⁺ and Zn²⁺ in the acidic pH side, and by Ca²⁺ and Mn²⁺ in the alkaline pH side.     

The Stabilizing Effects of O-glycosylation on the Secondary Structural Integrity of the Designed α-loop-α motif by Molecular Dynamics Simulations

Abstract

In this study, various 400 ps molecular dynamics simulations were conducted to determine the stabilizing effect of O-glycosylation on the secondary structural integrity of the design α-loop-α motif, which has the optimal loop length of 7 Gly residues (denoted as N-A₁₆G₇A₁₆-C). In general, O-glycosylation stabilizes the structural integrity of the model peptide regardless of the length and position of glycosylation sites because it decreases the opportunity for water molecules to compete for the intramolecular hydrogen bonds. The designed peptide exhibits the highest helicity when residues 11 and 31 are replaced with Ser residues followed by O-linked with 3 galactose residues, representing the “face-to-face” glycosylation near the loop. In this case, the loop exhibits an extended conformation and several new hydrogen bonds are observed between the main chain of the loop and the galactose residues, resulting in decreasing the fluctuation and increasing the stability of the entire peptide. When the glycosylation are made close to the loop, the secondary structural integrity of the α-loop-α motif increases with the number of galactose residues. In addition, “face- to-face” glycosylation increases the structural integrity of this motif to a greater extent than “back-to-back” glycosylation. However, when the glycosylation are created away from the loop and near the N- and C-termini, no general rule is found for the stabilizing effect.     

The relationship between visual working memory and attention: retention of precise colour information in the absence of effects on perceptual selection

We examined the conditions under which a feature value in visual working memory (VWM) recruits visual attention to matching stimuli. Previous work has suggested that VWM supports two qualitatively different states of representation: an active state that interacts with perceptual selection and a passive (or accessory) state that does not. An alternative hypothesis is that VWM supports a single form of representation, with the precision of feature memory controlling whether or not the representation interacts with perceptual selection. The results of three experiments supported the dual-state hypothesis. We established conditions under which participants retained a relatively precise representation of a parcticular colour. If the colour was immediately task relevant, it reliably recruited attention to matching stimuli. However, if the colour was not immediately task relevant, it failed to interact with perceptual selection. Feature maintenance in VWM is not necessarily equivalent with feature-based attentional selection.     

Evolution of temperature responses in the Cladophora vagabunda complex and the C. albida/sericea complex (Chlorophyta)

Differentiation in temperature responses (survival and growth) was investigated among isolates of two tropical to temperate green algal lineages: the Cladophora vagabunda complex and the C. albida/sericea complex. The results were analysed in relation to published data on 18S rRNA and ITS sequence divergence, which have shown that the overall degree of genetic divergence is similar in the two lineages but that very different patterns of radiation have occurred. In the C. vagabunda complex, the two main clades in the well-resolved phylogenetic tree differed mainly in their tolerance to low temperatures. Within-clade variation was no stronger in the Atlantic/Pacific than in the all-Pacific clade. In the C. albida/sericea complex, six distinctive ITS types indicated early radiation. Although distinctive differences were found between some of these types, the thermal responses of others were very similar, indicating physiological stasis. In both lineages there was evidence for some adaptation to local temperature regimes but phylogenetic constraints were generally more important. Isolates with the same ITS sequences showed similar temperature responses even though collected from different climate zones. Evidence was found for a physiological trade-off between growth at high and at low temperatures in the C. albida/sericea complex, whereas, in the C. vagabunda complex, one clade showed more eurythermal growth responses than the other. In the C. vagabunda complex, which is the ancestral lineage of the C. albida/sericea complex, major differentiation was found in cold tolerance but not in heat tolerance, whereas the reverse pattern was found in the derived C. albida/sericea complex. These findings suggest that an acquisition of cold tolerance preceded the loss of heat tolerance during adaptation to colder climates.