Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Electrically-Evoked Frequency-Following Response (EFFR) in the Auditory Brainstem of Guinea Pigs

It is still a difficult clinical issue to decide whether a patient is a suitable candidate for a cochlear implant and to plan postoperative rehabilitation, especially for some special cases, such as auditory neuropathy. A partial solution to these problems is to preoperatively evaluate the functional integrity of the auditory neural pathways. For evaluating the strength of phase-locking of auditory neurons, which was not reflected in previous methods using electrically evoked auditory brainstem response (EABR), a new method for recording phase-locking related auditory responses to electrical stimulation, called the electrically evoked frequency-following response (EFFR), was developed and evaluated using guinea pigs. The main objective was to assess feasibility of the method by testing whether the recorded signals reflected auditory neural responses or artifacts. The results showed the following: 1) the recorded signals were evoked by neuron responses rather than by artifact; 2) responses evoked by periodic signals were significantly higher than those evoked by the white noise; 3) the latency of the responses fell in the expected range; 4) the responses decreased significantly after death of the guinea pigs; and 5) the responses decreased significantly when the animal was replaced by an electrical resistance. All of these results suggest the method was valid. Recording obtained using complex tones with a missing fundamental component and using pure tones with various frequencies were consistent with those obtained using acoustic stimulation in previous studies.     

Effects of SARA on Oxygen–Glucose Deprivation in PC12 Cell Line

Ischemic stroke is a major composition of cerebrovascular disease, seriously threatening to human health in the world. Activin A (ActA), belonging to transforming growth factor-beta (TGF-β) super family, plays an important role in the hypoxic-ischemic brain injury through ActA/Smads pathway. While as an essential phosphorylation assistor in TGF-β signaling, the functions and mechanisms of smad anchor for receptor activation (SARA) in ischemic brain injury remain poorly understood. To solve this problem and explore the pathological processes of ischemic stroke, we used an Oxygen–Glucose deprivation (OGD) model in nerve growth factor-induced differentiated rattus PC12 pheochromocytoma cells and down regulated the expressions of SARA by RNA interference technology. Our results showed that the repression of SARA before OGD exposure reduced the expressions of Smad2, 3, 4 mRNA and the phosphorylation rate of Smad2 protein, but it did not affect the mRNA expressions of Smad7. After OGD treatment, ActA/Smads pathway was activated and the expression of SARA in the SARA pre-repression group was significantly up-regulated. The pre-repression of SARA increased the sensitivities of nerve-like cells to OGD damage. Moreover, the mRNA expression of Smad7 which was supposed to participate in the negative feedback of ActA/Smads pathway was also elevated due to OGD injury. Taken together, these results suggest a positive role of SARA in assisting the phosphorylation of Smad2 and maintaining the neuron protective effect of ActA/Smads pathway.     

Induced leaf intercellular CO2, photosynthesis, potassium and nitrate retention and strawberry early fruit formation under macronutrient limitation

Relationships between induced high leaf intercellular CO₂ concentrations, leaf K⁺ and NO₃ ^? ion movement and early fruit formation under macronutrient limitation are not well understood. We examined the effects and interactions of reduced K/N input treatments on leaf intercellular CO₂, photosynthesis rate, carboxylation and water use efficiency, berry formation as well as leaf/fruit K⁺, NO₃ ^? and photosynthate retention of strawberry (Fragaria × ananassa Duch.) to enhance low-input agriculture. The field study was conducted in Nova Scotia, eastern Canada during 2009–2010. The experimental treatments consisted of five K₂O rates (0, 6, 12, 18, and 24 kg ha^?1) and five N rates (0, 5, 10, 15, and 20 kg ha^?1), representing respectively, 0, 25, 50, 75, and 100 % of regular macronutrient recommendations based on the soil testing. The treatments were arranged in a split-plot design with three blocks in the field. The cultivar was ‘Mira’, a June-bearing crop. The results showed that strawberry plants treated with 25 %-reduced inputs could induce significantly higher leaf intercellular CO₂ concentrations to improve plant photosynthesis, carboxylation and water use efficiency and translocation of leaf/fruit K⁺ and dissolved solids, which could advance berry formation by 6 days and produce significantly higher marketable yields (P < 0.05). Higher leaf intercellular CO₂ inhibited leaf/fruit NO₃ ^? ion retention, but this inhibition did not occur in leaf/fruit K⁺ retention. Linear interactions of the K/N treatments were significant on fruit marketable yields, intercellular CO₂, net photosynthesis, leaf transpiration rates, and leaf temperatures (P < 0.05). It was concluded that higher leaf CO₂ could enhance plant photosynthesis, promote plant carboxylation and water use efficiency, and advance berry formation, but it could inhibit leaf NO₃ ^? retention. This inhibition did not find in leaf K⁺ ion and dissolved solid retention. Overlay co-limitation of leaf intercellular CO₂ and translocation of leaf/fruit K⁺/NO₃ ^? and total dissolved solids could constrain more fruit formation attributes under full macronutrient supply than reduced inputs. It was suggested that low input would be an optimal and sustainable option for improving small fruit crop physiological development and dealing with macronutrient deficiency challenge.     

Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc1 complex

The homodimeric bc₁ complexes are membrane proteins essential in respiration and photosynthesis. The ~ 11 Å distance between the two b_L-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc₁ complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer. Selection pressure under photosynthetic growth always favored the homodimer over heterodimeric variants enforcing inter-monomer electron transfer, showing that the latter are not competitive. These results, together with kinetic analysis of myxothiazol titrations, demonstrate that inter-monomer electron transfer does not occur at rates competitive with monomeric turnover. We examine the results from other groups interpreted as demonstrating rapid inter-monomer electron transfer, conclude that similar mechanisms are likely to be in play, and suggest that such claims might need to be re-examined.     

Construction and Characterization of a CTLA-4-Targeted scFv–Melittin Fusion Protein as a Potential Immunosuppressive Agent for Organ Transplant

Immunotoxins with selective cytotoxicity are frequently used as therapeutic immunosuppressive agents in solid-organ transplantation because of their efficiency and high specificity. In this study, we present a new recombinant immunotoxin termed anti-CTLA-4-scFv–melittin prepared from Escherichia coli aimed at clearing activated T cells at the same time avoiding all-round decline in systematic immunity. This fusion protein is composed of anti-CTLA-4-scFv unit and melittin analog unit with properties of low immunogenicity and selective cytotoxicity to CTLA-4-positive T cells. In preliminary biological activity assays, our results confirmed the feasibility of activated T cell clearance strategy and there were significant differences in cell survival rates between CTLA-4-positive group and control group at all experimental concentrations of the immunotoxin. The selective cytotoxicity, low immunogenicity, and low production cost make it an attractive alternate to traditional immunosuppressants.     

Albino inflorescence proliferation of Dendrocalamus latiflorus

Summary Inflorescence proliferation is a plant tissue culture technique that, can be used to obtain in vitro inflorescences year-round without the intervening development of vegetative organs. In this study, we used albino mutant inflorescences of Dendrocalamus latiflorus as the original explant material to investigate, the effect of plant growth regulators on long-term inflorescence proliferation. The albino inflorescences proliferated on solidified Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), and the optimal concentration for successful long-term inflorescence proliferation was 0.45 μM TDZ. A combination of α-naphthaleneacetic acid (NAA) with 0.45 μM TDZ inhibited the inflorescence proliferation. Inflorescences cultured on a TDZ-free medium supplemented with 26.82 μM NAA rooted in 21 d, vegetative shoots formed by 42 d and, in one case, flowering occurred after 63 d. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D, 4.52 μM) and pieloram (4.14 μM) induced shoot formation. The protocol described can be used to produce large numbers of mutant inflorescences within a relatively short period of time.     

Motor-Substrate Interactions in Mycoplasma Motility Explains Non-Arrhenius Temperature Dependence

Mycoplasmas exhibit a novel, substrate-dependent gliding motility that is driven by ∼400 “leg” proteins. The legs interact with the substrate and transmit the forces generated by an assembly of ATPase motors. The velocity of the cell increases linearly by nearly 10-fold over a narrow temperature range of 10-40°C. This corresponds to an Arrhenius factor that decreases from ∼45 k_BT at 10°C to ∼10 k_BT at 40°C. On the other hand, load-velocity curves at different temperatures extrapolate to nearly the same stall force, suggesting a temperature-insensitive force-generation mechanism near stall. In this article, we propose a leg-substrate interaction mechanism that explains the intriguing temperature sensitivity of this motility. The large Arrhenius factor at low temperature comes about from the addition of many smaller energy barriers arising from many substrate-binding sites at the distal end of the leg protein. The Arrhenius dependence attenuates at high temperature due to two factors: 1), the reduced effective multiplicity of energy barriers intrinsic to the multiple-site binding mechanism; and 2), the temperature-sensitive weakly facilitated leg release that curtails the power stroke. The model suggests an explanation for the similar steep, sub-Arrhenius temperature-velocity curves observed in many molecular motors, such as kinesin and myosin, wherein the temperature behavior is dominated not by the catalytic biochemistry, but by the motor-substrate interaction.