The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

6-Phosphofructo-2-kinase and D-fructose-2,6-bisphosphatase activities in mung bean seedlings

6-Phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase) and D-fructose-2,6-bisphosphatase activities have been found in extracts prepared from etiolated mung bean seedlings. The activity of 6-phosphofructo-2-kinase exhibits a sigmoidal shape in response to changes in concentrations of both substrates, D-fructose 6-phosphate and ATP (S_0.5 values of 1.8 and 1.2 mM, respectively). Inorganic orthophosphate (P_i) has a strong stimulating effect on the 2-kinase activity (A_0.5 at about 2 mM), moderately increasing the V_max and modifying the response into hyperbolic curves with K_m values of 0.4 and 0.2 mM for fructose 6-phosphate and ATP, respectively. 3-Phosphoglycerate (I_0.5 about 0.15 mM) partially inhibited the kinase activity by counteracting the P_i activation. In contrast, the activity of D-fructose-2,6-bisphosphatase (K_m 0.38 mM) is strongly inhibited by P_i (I_0.5 0.8 mM) lowering its affinity to fructose-2,6-P₂ (K_m 1.4 mM). 3-Phosphoglycerate activites the enzyme (A_0.5 at about 0.3 mM) without causing a significant change in its K_m for fructose-2,6-P₂. The activities of both of these enzymes in relationship to the metabolic role of D-fructose 2,6-bisphosphate in the germinating seed is discussed.     

ACE/ACE2 Ratio and MMP-9 Activity as Potential Biomarkers in Tuberculous Pleural Effusions

Objective: Pleural effusion is common problem, but the rapid and reliable diagnosis for specific pathogenic effusions are lacking. This study aimed to identify the diagnosis based on clinical variables to differentiate pleural tuberculous exudates from other pleural effusions. We also investigated the role of renin-angiotensin system (RAS) and matrix metalloproteinase (MMPs) in the pathogenesis of pleural exudates.Experimental design: The major components in RAS and extracellular matrix metabolism, including angiotensin converting enzyme (ACE), ACE2, MMP-2 and MMP-9 activities, were measured and compared in the patients with transudative (n = 45) and exudative (n = 80) effusions. The exudative effusions were come from the patients with tuberculosis (n = 20), pneumonia (n = 32), and adenocarcinoma (n = 28).Results: Increased ACE and equivalent ACE2 activities, resulting in a significantly increased ACE/ACE2 ratio in exudates, were detected compared to these values in transudates. MMP-9 activity in exudates was significantly higher than that in transudates. The significant correlation between ACE and ACE2 activity that was found in transudates was not found in exudates. Advanced analyses showed significantly increased ACE and MMP-9 activities, and decreased ACE2 activity in tuberculous pleural effusions compared with those in pneumonia and adenocarcinoma effusions. The results indicate that increased ACE and MMP-9 activities found in the exudates were mainly contributed from a higher level of both enzyme activities in the tuberculous pleural effusions.Conclusion: Interplay between ACE and ACE2, essential functions in the RAS, and abnormal regulation of MMP-9 probably play a pivotal role in the development of exudative effusions. Moreover, the ACE/ACE2 ratio combined with MMP-9 activity in pleural fluid may be potential biomarkers for diagnosing tuberculous pleurisy.     

神经递质和调质参与导水管周围灰质痛觉调控的研究进展

导水管周围灰质(periaqueductal gray,PAG)在疼痛的调控过程中处于一个不可或缺的位置.其不仅是痛觉信息上行传递的重要部位,还是疼痛抑制系统的重要组成部分.在PAG,包括γ-氨基丁酸(γ-aminobutyric acid,GABA)、5-羟色胺(5-hydroxytryptamine,5-HT)和谷氨酸(glutamate,Glu)在内的神经递质以及内源性阿片肽(endogenous opioid peptides,EOP)和内源性大麻素(endocannabinoid,e CB)为代表的神经调质都参与了PAG对疼痛的信息传递以及调节.本文重点综述GABA、5-HT、Glu、EOP和eCB在PAG参与疼痛生理调控机制的研究进展,以期为中枢神经系统的镇痛研究提供一定的理论基础.     

Infradian Rhythmicity in Sleep/Wake Ratio in Developing Infants

Although there are several reports on ultradian and circadian rhythms in newborns, we found only one report in which infradian periodicities are described for heart-rate measurements in the early stages of human development. Here, we report infradian rhythms in the monthly range in the sleep/wake cycle of four infants studied along 24 consecutive weeks. Our procedure was applied to sleep diary records from four healthy newborns. The data were arranged in binary time series representing sleep (?1) or wake (1) states. These time series were integrated in order to obtain the cumulative sleep/wake time. A measure of the sleep/wake ratio (SWR) was obtained by computing the average slope of the cumulative sleep/wake time. To extract periodicities we applied the Fourier periodogram to the temporal course of the SWR. We found a notorious difference in the SWR pattern among infants. In two infants the SWR showed a marked linear decay, spending more time asleep than awake, while in the two other infants oscillated near zero. We found robust oscillations in all children. In all cases the Fourier periodogram results present significant power in the infradian range. From these results, we suggest that sleep and wake durations are probably modulated by some internal stimuli.     

Production of acidic xylo-oligosaccharides by a family 10 endoxylanase from Thermoascus aurantiacus and use as plant growth regulators

Neutral and acidic oligosaccharides were obtained from birchwood xylan by treatment with an endoxylanase, family 10 class, from Thermoascus aurantiacus. The main acidic xylooligosaccharide (aldotetrauronic acid) was separated from the hydrolysate by anion-exchange and size-exclusion chromatography and the primary structure was determined by ¹³C NMR spectroscopy. The aldotetrauronic yield was 15% (w/w) of the total solubilised sugars. The addition of purified aldoterauronic acid at 1.6–16 mg l^–1 growth medium, induced callus and somatic embryogenesis in culture explants of common mallow (Malva silvestris L.) and cotton (Gosssypium hirsutum).     

In vitro binding to and in vivo effect on the cytosol and nuclear progesterone receptors of various progestins,and their relationship to synthesis of uteroglobin in rabbit uterus

In vitro binding affinities of various progestins to cytosol and nuclear progesterone receptors of rabbit uterus were determined and correlated with the biological potency of these steroids. In addition, cytosol and nuclear progesterone receptor levels were measured after a 5-day administration of different progestins (0.5 mg/kg daily) with variable biologic activites. The receptor levels were compared with the bilological response; the induction of uteroglobin synthesis. Cytosol and nuclear progesterone receptors had identical steroid binding properties (r = 0.98). The correlation between the in vitro binding affinity (cytosol or nuclear) and the in vivo biologic activity of the steroids was good (r = 0.73). After a 5-day treatment with progestins, the nuclear receptor concentration correlated in an inverse manner (r = ?0.84) with the uterine fluid unteroglobin concentration. A similar, but slightly weaker correlation (r = ?0.81) was also found for the cytosol receptor content and uteroglobin secretion. These data indicate that not only nuclear, but also cytosol progesterone receptor levels decrease in the rabbit uterus during chronic hormone action. Decline in the nuclear progesterone receptor content seemed to occur during treatment with all progestational steroids, while onlyi progestins with high biological potency were capable of decreasing the cytosol receptor content.     

The CD38/NAD/SIRTUIN1/EZH2 Axis Mitigates Cytotoxic CD8 T Cell Function and Identifies Patients with SLE Prone to Infections

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Absolute configuration and conformation of the pure opioid antagonist (+)-2,9 alpha-dimethyl-5-(m-hydroxyphenyl)morphan.

(+)-2,9 alpha-Dimethyl-5-(m-hydroxyphenyl)morphan is the only phenylmorphan analog whose affinity for opioid kappa-receptors is greater than its affinity for opioid mu-receptors. Pharmacologically, the compound is a pure opioid antagonist devoid of agonist activity in in vivo assays of antinociception. The absolute configuration of the compound has been determined to be (1R,5S,9R) from an X-ray crystallographic study of the chloride salt. Thus, the absolute configuration corresponds to that of the atypical opioid agonist (-)-phenylmorphan while the weak atypical agonist (-)-2,9 alpha-dimethyl-5-(m- hydroxyphenyl)morphan corresponds to the potent morphine-like (+)-phenylmorphan. The preferred orientations of the phenyl ring for the two stereoisomers were determined using the molecular mechanics program MM2-87 and found to vary from that of the two parent compounds. The atypical properties of the two 9 alpha-methyl analogs is consistent with an opioid ligand model which proposes that morphine-like properties require a particular range of phenyl orientations. There was good agreement between the structure obtained from X-ray crystallography and computed with the MM2-87 program.