Structural insights into unique substrate selectivity of Thermoplasma acidophilum D-aldohexose dehydrogenase

The d-aldohexose dehydrogenase from the thermoacidophilic archaea Thermoplasma acidophilum (AldT) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and catalyzes the oxidation of several monosaccharides with a preference for NAD⁺ rather than NADP⁺ as a cofactor. It has been found that AldT is a unique enzyme that exhibits the highest dehydrogenase activity against d-mannose. Here, we describe the crystal structures of AldT in ligand-free form, in complex with NADH, and in complex with the substrate d-mannose, at 2.1 Å, 1.65 Å, and 1.6 Å resolution, respectively. The AldT subunit forms a typical SDR fold with an unexpectedly long C-terminal tail and assembles into an intertwined tetramer. The d-mannose complex structure reveals that Glu84 interacts with the axial C2 hydroxyl group of the bound d-mannose. Structural comparison with Bacillus megaterium glucose dehydrogenase (BmGlcDH) suggests that the conformation of the glutamate side-chain is crucial for discrimination between d-mannose and its C2 epimer d-glucose, and the conformation of the glutamate side-chain depends on the spatial arrangement of nearby hydrophobic residues that do not directly interact with the substrate. Elucidation of the d-mannose recognition mechanism of AldT further provides structural insights into the unique substrate selectivity of AldT. Finally, we show that the extended C-terminal tail completely shuts the substrate-binding pocket of the neighboring subunit both in the presence and absence of substrate. The elaborate inter-subunit interactions between the C-terminal tail and the entrance of the substrate-binding pocket imply that the tail may play a pivotal role in the enzyme activity.     

Postoperative radiotherapy in prostate cancer: Analysis of prognostic factors in a series of 282 patients

Aim

To assess the outcomes of patients treated with postoperative RT in relation to the possible prognostic factors.

Background

Postoperative radiotherapy (RT) has been proved to reduce the risk of biochemical recurrence in high-risk prostate cancer patients. Baseline prostate specific antigen (PSA), pathological Gleason score (GS), positive surgical margins, nodal status and seminal vesicle invasion are independent predictors of biochemical relapse.

Materials and methods

The clinical records of 282 patients who underwent postoperative RT were retrospectively reviewed. The prognostic value of postoperative PSA, preoperative risk class, nodal status, pathological GS, margins status, and administration of hormonal therapy (HT) was analyzed.

Results

Postoperative RT was delivered with a median dose to the prostatic fossa of 66 Gy (range 50–72) in 1.8–2 Gy/fraction. Median follow-up was 23.1 months (range 6–119). Five-year actuarial biochemical disease-free survival (bDFS) and overall survival rates were 76% and 95%, respectively. Higher bDFS was found for patients with postoperative PSA <0.02 ng/ml (p = 0.03), low preoperative risk class (p = 0.01), pN0 (p = 0.003), GS 4–6 (p = 0.0006), no androgen deprivation therapy (p = 0.02), and irrespective of surgical margin status (p = 0.10). Multivariate analysis showed that postoperative PSA and Gleason score had a significant impact on bDFS (p = 0.039 and p = 0.05, respectively).

Conclusions

Postoperative RT with a dose of 66 Gy offers an acceptable toxicity and an optimal disease control after radical prostatectomy in patients with different risk features. A postoperative PSA >0.02 ng/ml could be considered as a prognostic factor and a tool to select patients at risk for progression.     

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Detection of nanometer-sized particles in living cells using modern fluorescence fluctuation methods

Nanosized materials are increasingly used in medicine and biotechnology but originate also from various aerosol sources. A detailed understanding of their interaction with cells is a prerequisite for specific applications and appraisal of hazardous effects. Fluorescence fluctuation methods are applied to follow the time-course of the translocation and distribution of fluorescent 20 nm polystyrene nanoparticles with negative surface charges in HeLa cells under almost physiological conditions. The experimental results demonstrate that singular particles enter the cell without significant contribution by endocytotic mechanisms and are distributed within the cytoplasm. Subsequently aggregation is observed, which can be blocked by cytotoxins, like Genistein and Cytochalasin B, interfering with cellular uptake processes. The observed non-active uptake is due to non-specific interactions with the cell surface and could be responsible for distribution of nanometer-sized materials in tissue.     

Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro

Small interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limited because of in vivo instability. As a first step to overcome delivery issues, a series of graft copolymers of polyethylene glycol and polyethylenimine (PEI-g-PEG) were synthesized and investigated as nontoxic carriers for delivery of siRNA targeting the signaling peptide of secretory clusterin (sCLU), a prosurvival factor that protects cells from ionizing radiation (IR) injury, as well as chemotherapeutic agents. Three copolymers with different PEG grafting densities were tested for their abilities to bind and form nanocomplexes with siRNA. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2k10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and had the smallest nanocomplex size (115 +/- 13 nm diameter). In human breast cancer MCF-7 cells, 2k10-siRNA-sCLU nanocomplexes suppressed both basal as well as IR-induced sCLU protein expression, which led to an over 3-fold increase in IR-induced lethality over 2k10-siRNA scrambled controls. In summary, this study demonstrates the proof-of-principle in using nanoparticle-mediated delivery of specific siRNAs to enhance the lethality of IR exposure in vitro, opening the door for siRNA-mediated knockdown of specific cytoprotective factors, such as DNA repair, anti-apoptotic, free radical scavenging, and many other proteins.     

Quality of life and radiotherapy in brain metastasis patients

AimThe primary objective of this study was to assess whether there was an improvement in QoL for patients with brain metastases after radiotherapy treatments.BackgroundAssessment of quality of life (QoL) in brain metastasis patients has become increasingly recognized as an important outcome.Materials and methodsPatients treated for brain metastasis in our department during 2010 were included in our prospective study. QoL assessments were conducted at baseline, 1 month, and 3 months after completion of whole-brain radiotherapy (WBRT). Wilcoxon test for multiple comparisons was calculated to detect significant differences in global QoL scores.ResultsThirty-nine patients with brain metastases completed the EORTC QLQ-C30/BN-20 questionnaire independently. Median age was 59.9 years (from 37 to 81 years). Our results report differences between the baseline and 3 months in worsening of a global health status (p = 0.034) and cognitive function (p = 0.004), as well as drowsiness (p = 0.001), appetite loss (p = 0.031) and hair loss (p = 0.005). There is a tendency for deterioration of physical function (p = 0.004), communication deficit (p = 0.012), and weakness of legs (p = 0.024), between the baseline and 1 month evaluation. There was no difference in a global cognitive status between different evaluations. Median survival time was 3 months (CI 95% 1.85; 4.15).ConclusionsOur findings indicate a small deterioration for a global QoL status, and large deterioration for cognitive function after radiation treatments, as well as worsening of brain metastasis related symptom items. Further research is necessary to refine treatment selection for patients with brain metastases, since it may at least contribute to the stabilization of their QoL status.     

A modified approach to the measurement problem: objective reduction in the retinal molecule prior to conformational change

A new analysis of the measurement problem reveals the possibility that collapse of the wavefunction may now take place just before photoisomerization of the rhodopsin molecule in the retinal rods. It is known that when a photon is initially absorbed by the retinal molecule which, along with opsin comprises the rhodopsin molecule, an electron in the highest pi orbital is immediately excited to a pi* orbital. This means that a measurement or transfer of information takes place at the quantum level before the retinal molecule commences the conformational change from cis to trans. This could have profound implications for resolving some of the foundational issues confronting quantum mechanics.     

参棚透光率对西洋参叶片光合作用的影响

研究了参棚透光率与西洋参叶片净光合速率之间的关系.结果表明,西洋参叶片光饱和点、净光合速率及其日变型均随参棚透光率的不同,存在一定的差异.4年生西洋参叶片在12%、30%、42%3种透光率下(气温29.0℃左右),光饱和点分别为171.0、323.0和429.0μmol·m^-2·s^-1,净光合速率最大值为6.54mg·dm^-2·h^-1(CO₂),出现在透光率为30%的参棚下;3年生西洋参在透光率不超过25.8%的参棚下,叶片净光合速率日变化呈单峰型,透光率大于25.8%时,呈双峰型,参叶“光合午休”现象明显.单相关分析表明,光量子通量密度是影响西洋参净光合速率的主要因子;回归分析结果表明,各影响因子对参叶净光合速率的综合影响显著.