DBSI: DNA-binding site identifier

In this study, we present the DNA-Binding Site Identifier (DBSI), a new structure-based method for predicting protein interaction sites for DNA binding. DBSI was trained and validated on a data set of 263 proteins (TRAIN-263), tested on an independent set of protein-DNA complexes (TEST-206) and data sets of 29 unbound (APO-29) and 30 bound (HOLO-30) protein structures distinct from the training data. We computed 480 candidate features for identifying protein residues that bind DNA, including new features that capture the electrostatic microenvironment within shells near the protein surface. Our iterative feature selection process identified features important in other models, as well as features unique to the DBSI model, such as a banded electrostatic feature with spatial separation comparable with the canonical width of the DNA minor groove. Validations and comparisons with established methods using a range of performance metrics clearly demonstrate the predictive advantage of DBSI, and its comparable performance on unbound (APO-29) and bound (HOLO-30) conformations demonstrates robustness to binding-induced protein conformational changes. Finally, we offer our feature data table to others for integration into their own models or for testing improved feature selection and model training strategies based on DBSI.     

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.     

Specificity of extrafloral nectar induction by herbivores differs among native and invasive populations of tallow tree

Background and Aims

Invasive plants can be released from specialist herbivores and encounter novel generalists in their introduced ranges, leading to variation in defence among native and invasive populations. However, few studies have examined how constitutive and induced indirect defences change during plant invasion, especially during the juvenile stage.

Methods

Constitutive extrafloral nectar (EFN) production of native and invasive populations of juvenile tallow tree (Triadica sebifera) were compared, and leaf clipping, and damage by a native specialist (Noctuid) and two native generalist caterpillars (Noctuid and Limacodid) were used to examine inducible EFN production.

Key results

Plants from introduced populations had more leaves producing constitutive EFN than did native populations, but the content of soluble solids of EFN did not differ. Herbivores induced EFN production more than simulated herbivory. The specialist (Noctuid) induced more EFN than either generalist for native populations. The content of soluble solids in EFN was higher (2·1 times), with the specialist vs. the generalists causing the stronger response for native populations, but the specialist response was always comparable with the generalist responses for invasive populations.

Conclusions

These results suggest that constitutive and induced indirect defences are retained in juvenile plants of invasive populations even during plant establishment, perhaps due to generalist herbivory in the introduced range. However, responses specific to a specialist herbivore may be reduced in the introduced range where specialists are absent. This decreased defence may benefit specialist insects that are introduced for classical biological control of invasive plants.     

Auxin distribution is differentially affected by nitrate in roots of two rice cultivars differing in responsiveness to nitrogen

Background and Aims

Although ammonium (NH₄⁺) is the preferred form of nitrogen over nitrate (NO₃⁻) for rice (Oryza sativa), lateral root (LR) growth in roots is enhanced by partial NO₃⁻ nutrition (PNN). The roles of auxin distribution and polar transport in LR formation in response to localized NO₃⁻ availability are not known.

Methods

Time-course studies in a split-root experimental system were used to investigate LR development patterns, auxin distribution, polar auxin transport and expression of auxin transporter genes in LR zones in response to localized PNN in ‘Nanguang’ and ‘Elio’ rice cultivars, which show high and low responsiveness to NO₃⁻, respectively. Patterns of auxin distribution and the effects of polar auxin transport inhibitors were also examined in DR5::GUS transgenic plants.

Key Results

Initiation of LRs was enhanced by PNN after 7 d cultivation in ‘Nanguang’ but not in ‘Elio’. Auxin concentration in the roots of ‘Nanguang’ increased by approx. 24 % after 5 d cultivation with PNN compared with NH₄⁺ as the sole nitrogen source, but no difference was observed in ‘Elio’. More auxin flux into the LR zone in ‘Nanguang’ roots was observed in response to NO₃⁻ compared with NH₄⁺ treatment. A greater number of auxin influx and efflux transporter genes showed increased expression in the LR zone in response to PNN in ‘Nanguang’ than in ‘Elio’.

Conclusions

The results indicate that higher NO₃⁻ responsiveness is associated with greater auxin accumulation in the LR zone and is strongly related to a higher rate of LR initiation in the cultivar ‘Nanguang’.     

Sinomenine hydrochloride enhancement of the inhibitory effects of anti-transferrin receptor antibody-dependent on the COX-2 pathway in human hepatoma cells

Transferrin receptor (TfR) has been used as a target for the antibody-based therapy of cancer due to its higher expression in tumors relative to normal tissues. Great potential has been shown by anti-TfR antibodies combined with chemotherapeutic drugs as a possible cancer therapeutic strategy. In our study, we investigated the anti-tumor effects of anti-TfR monoclonal antibody (mAb) alone or in combination with sinomenine hydrochloride in vitro. Results suggested that anti-TfR mAb or sinomenine hydrochloride could induce apoptosis, inhibit proliferation, and affect the cell cycle. A synergistic effect was found in relation to tumor growth inhibition and the induction of apoptosis when anti-TfR mAb and sinomenine hydrochloride were used simultaneously. The expression of COX-2 and VEGF protein in HepG2 cells treated with anti-TfR mAb alone was increased in line with increasing dosage of the agent. In contrast, COX-2 expression was dramatically decreased in HepG2 cells treated with sinomenine hydrochloride alone. Furthermore, we demonstrated that the inhibitory effects of sinomenine hydrochloride and anti-TfR mAb administered in combination were more prominent than when the agents were administered singly. To sum up, these results showed that the combined use of sinomenine hydrochloride and anti-TfR mAb may exert synergistic inhibitory effects on human hepatoma HepG2 cells in a COX-2-dependent manner. This finding provides new insight into how tumor cells overcome the interference of iron intake to survive and forms the basis of a new therapeutic strategy involving the development of anti-TfR mAb combined with sinomenine hydrochloride for liver cancer.     

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.     

The Role of HSPA12B in Regulating Neuronal Apoptosis

Heat shock protein A12B (HSPA12B) is the newest member of a recently defined subfamily of proteins distantly related to the 70-kDa family of heat shock proteins (HSP70) family. HSP70s play a crucial role in protecting cells, tissues, organs and animals from various noxious conditions. Here we studied the dynamic expression changes and localization of HSPA12B after middle cerebral artery occlusion (MCAO) with reperfusion induced ischemic insult processes in adult rats. Apoptosis, as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, was also increased in the peri-ischemic cortex compared to non-ischemic hemisphere. The expression of HSPA12B was strongly induced in the ischemic hemisphere of MCAO reperfusion rats in vivo. In vitro studies indicated that the up-regulation of HSPA12B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knockdown of HSPA12B in cultured differentiated PC12 cells by siRNA showed that HSPA12B inhibited the expression of active caspase-3. Collectively, these results suggested that HSPA12B may be required for protecting neurons from ischemic insults.