酶、多肽电荷数的理论计算及误差研究

对酶的电荷数随ｐＨ变化的定量关系进行了理论推导,将推导出的酶的电荷数与ｐＨ的关系式应用于多肽等电点的计算,理论计算结果与文献实验结果完全一致,并推导出酸性氨基酸、碱性氨基酸及中性氨基酸等电点的计算式,与现有计算式完全一致．应用荧光光谱和荧光偏振对肌酸激酶带电性随ｐＨ值的变化关系的研究表明,理论计算符合实验结果,表明：此文理论关系式是可靠的．     

Lupane‐ and Friedelane‐Type Triterpenoids from Celastrus stylosus

Two new triterpenoids, 30‐hydroxylup‐20(29)‐ene 3β‐caffeate ( 1 ) and 24‐nor‐friedelan‐6α,10‐dihydroxy‐1,2‐dioxo‐4,7‐dien‐29‐oic acid ( 2 ), together with eight known compounds 3 – 10 , were isolated from the roots of Celastrus stylosus. The structures of these compounds were elucidated on the basis of spectroscopic analyses. To the best of our knowledge, this represents the first study on the chemical constituents of C. stylosus. The antiproliferative activities of the triterpenoids against six human cancer cell lines (PANC‐1, A549, PC‐3, HepG2, SGC‐7901, and HCCLM3) were evaluated. Compounds 3, 4 , and 10 exhibited comparable activities against PC‐3 and HCCLM3 cell lines as the positive control taxol.     

Evaluation of 111In-Labelled Exendin-4 Derivatives Containing Different Meprin β-Specific Cleavable Linkers

Background

Cleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.

Results

The insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the ¹¹¹In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound ¹¹¹In-Ex4NOD40.

Conclusion

In conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.     

Urinary Biomarkers at Early ADPKD Disease Stage

Background

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by a decline in renal function at late disease stage when the majority of functional renal parenchyma is replaced by cystic tissue. Thus, kidney function, assessed by estimated glomerular filtration rate (eGFR) does not well represent disease burden in early disease. Here, we investigated various urinary markers for tubular injury and their association with disease burden in ADPKD patients at early disease course.

Methods

ADPKD patients between 18 and 40 years with an eGFR greater or equal to 70 ml per min per 1.73m² were eligible for this cross-sectional study. Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL), Kidney Injury Molecule-1 (KIM-1), and Uromodulin (UMOD) were investigated by Enzyme-Linked Immunosorbent Assay. Clara Cell Protein 16 (CC16) was investigated by Latex Immuno Assay. Cryoscopy was performed to assess urine osmolality and Urinary Albumin-to-Creatinine Ratio (UACR) was calculated. The association and the predictive properties of the markers on eGFR and height adjusted total kidney volume (htTKV) was evaluated using multiple regression analysis, incorporating different control variables for adjustment. Internal bootstrapping validated the obtained results.

Results

In 139 ADPKD patients (age 31 ±7 years, mean eGFR of 93 ± 19 ml per min per 1.73 m²) the total kidney volume was negatively correlated with eGFR and UMOD and positive associated with age, UACR, KIM-1 and urine osmolality after adjustment for possible confounders. Urine osmolality and htTKV were also associated with eGFR, whereas no association of CC16, NGAL and UMOD with eGFR or htTKV was found.

Conclusion

UACR and urinary KIM-1 are independently associated with kidney size but not with renal function in our study population. Urine osmolality was associated with eGFR and kidney volume following adjustment for multiple confounders. Despite statistical significance, the clinical value of our results is not yet conceivable. Further studies are needed to evaluate the property of the aforementioned biomarkers to assess disease state at early ADPKD stage.     

Effect of Vegetation Rehabilitation on Soil Carbon and Its Fractions in Mu Us Desert,Northwest China

Although vegetation rehabilitation on semi-arid and arid regions may enhance soil carbon sequestration, its effects on soil carbon fractions remain uncertain. We carried out a study after planting Artemisia ordosica (AO, 17 years), Astragalus mongolicum (AM, 5 years), and Salix psammophila (SP, 16 years) on shifting sand land (SL) in the Mu Us Desert, northwest China. We measured total soil carbon (TSC) and its components, soil inorganic carbon (SIC) and soil organic carbon (SOC), as well as the light and heavy fractions within soil organic carbon (LF-SOC and HF-SOC), under the SL and shrublands at depths of 100 cm. TSC stock under SL was 27.6 Mg ha^?1, and vegetation rehabilitation remarkably elevated it by 40.6 Mgha^?1, 4.5 Mgha^?1, and 14.1 Mgha^?1 under AO, AM and SP land, respectively. Among the newly formed TSC under the three shrublands, SIC, LF-SOC and HF-SOC accounted for 75.0%, 10.7% and 13.1% for AO, respectively; they made up 37.0%, 50.7% and 10.6% for AM, respectively; they occupied 68.6%, 18.8% and 10.0% for SP, respectively. The accumulation rates of TSC within 0–100 cm reached 238.6 g m^?2y^?1, 89.9 g m^?2y^?1 and 87.9 g m^?2y^?1 under AO, AM and SP land, respectively. The present study proved that the accumulation of SIC considerably contributed to soil carbon sequestration, and vegetation rehabilitation on shifting sand land has a great potential for soil carbon sequestration.     

Maternal psychosocial stress during pregnancy alters the epigenetic signature of the glucocorticoid receptor gene promoter in their offspring: a meta-analysis

Prenatal stress has been widely associated with a number of short- and long-term pathological outcomes. Epigenetic mechanisms are thought to partially mediate these environmental insults into the fetal physiology. One of the main targets of developmental programming is the hypothalamic-pituitary-adrenal (HPA) axis as it is the main regulator of the stress response. Accordingly, an increasing number of researchers have recently focused on the putative association between DNA methylation at the glucocorticoid receptor gene (NR3C1) and prenatal stress, among other types of psychosocial stress. The current study aims to systematically review and meta-analyze the existing evidence linking several forms of prenatal stress with DNA methylation at the region 1_F of the NR3C1 gene. The inclusion of relevant articles allowed combining empirical evidence from 977 individuals by meta-analytic techniques, whose methylation assessments showed overlap across 5 consecutive CpG sites (GRCh37/hg19 chr5:142,783,607-142,783,639). From this information, methylation levels at CpG site 36 displayed a significant correlation to prenatal stress (r = 0.14, 95% CI: 0.05–0.23, P = 0.002). This result supports the proposed association between a specific CpG site located at the NR3C1 promoter and prenatal stress. Several confounders, such as gender, methylation at other glucocorticoid-related genes, and adjustment for pharmacological treatments during pregnancy, should be taken into account in further studies.     

On-Going Frontal Alpha Rhythms Are Dominant in Passive State and Desynchronize in Active State in Adult Gray Mouse Lemurs

The gray mouse lemur (Microcebus murinus) is considered a useful primate model for translational research. In the framework of IMI PharmaCog project (Grant Agreement n°115009, www.pharmacog.org), we tested the hypothesis that spectral electroencephalographic (EEG) markers of motor and locomotor activity in gray mouse lemurs reflect typical movement-related desynchronization of alpha rhythms (about 8–12 Hz) in humans. To this aim, EEG (bipolar electrodes in frontal cortex) and electromyographic (EMG; bipolar electrodes sutured in neck muscles) data were recorded in 13 male adult (about 3 years) lemurs. Artifact-free EEG segments during active state (gross movements, exploratory movements or locomotor activity) and awake passive state (no sleep) were selected on the basis of instrumental measures of animal behavior, and were used as an input for EEG power density analysis. Results showed a clear peak of EEG power density at alpha range (7–9 Hz) during passive state. During active state, there was a reduction in alpha power density (8–12 Hz) and an increase of power density at slow frequencies (1–4 Hz). Relative EMG activity was related to EEG power density at 2–4 Hz (positive correlation) and at 8–12 Hz (negative correlation). These results suggest for the first time that the primate gray mouse lemurs and humans may share basic neurophysiologic mechanisms of synchronization of frontal alpha rhythms in awake passive state and their desynchronization during motor and locomotor activity. These EEG markers may be an ideal experimental model for translational basic (motor science) and applied (pharmacological and non-pharmacological interventions) research in Neurophysiology.     

Application of Deamidated Gliadin Antibodies in the Follow-Up of Treated Celiac Disease

Introduction

The role of serological tests such as IgA anti-transglutaminase autoantibodies has become increasingly important in celiac disease (CD) diagnosis. However, the efficiency of these tests for patient follow-up is controversial. We investigated the correlation of 12 different serological tests, including recent deamidated gliadin and actin IgA tests, with villous atrophy (VA) in a retrospective cohort of treated celiac patients.

Materials and Methods

Serum samples were collected from 100 treated CD patients who had intestinal biopsy in the course of their follow-up. Antibodies against transglutaminase, deamidated gliadin peptides, and native gliadin were measured, along with IgA anti-actin. The biopsy slides were all blind-reviewed and scored according to Marsh classification.

Results

For all deamidated gliadin and transglutaminase tests, we found that a positive result was significantly associated with persistence of intestinal VA, with a diagnostic efficacy up to 80%. Furthermore, antibodies titers directly correlated with the degree of VA, indicating a strong link between disease activity and presence of antibodies in the serum. Interestingly, the tests with the highest association with persistent VA were those for deamidated gliadin IgG. Using a test positivity pattern analysis, we were also able to identify several groups of patients with distinct antibody profiles that showed significant differences in intestinal damage and diet compliance.

Conclusions

Altogether, these results show that deamidated gliadin antibodies are strongly correlated with VA and should be considered valuable tools in CD follow-up and that multiplex serologic analysis for treated CD represents a promising tool for personalized patient management.