MG53 protects against contrast-induced acute kidney injury by reducing cell membrane damage and apoptosis.

Mitsugumin 53 (MG53) is a tripartite motif family protein that has been reported to attenuate injury via membrane repair in different organs. Contrast-induced acute kidney injury (CI-AKI) is a common complication caused by the administration of iodinated contrast media (CM). While the cytotoxicity induced by CM leading to tubular cell death may be initiated by cell membrane damage, we wondered whether MG53 alleviates CI-AKI. This study was designed to investigate the effect of MG53 on CI-AKI and the underlying mechanism. A rat model of CI-AKI was established, and CI-AKI induced the translocation of MG53 from serum to injury sites on the renal proximal tubular (RPT) epithelia, as illustrated by immunoblot analysis and immunohistochemical staining. Moreover, pretreatment of rats with recombinant human MG53 protein (rhMG53, 2 mg/mL) alleviated iopromide-induced injury in the kidney, which was determined by measuring serum creatinine, blood urea nitrogen and renal histological changes. In vitro studies demonstrated that exposure of RPT cells to iopromide (20, 40, and 80 mg/mL) caused cell membrane injury and cell death, which were attenuated by rhMG53 (10 and 50 µg/mL). Mechanistically, MG53 translocated to the injury site on RPT cells and bound to phosphatidylserine to protect RPT cells from iopromide-induced injury. In conclusion, MG53 protects against CI-AKI through cell membrane repair and reducing cell apoptosis; therefore, rhMG53 might be a potential effective means to treat or prevent CI-AKI.

[Analysis of difficult problems on European Union laws and regulations of traditional herbal medicinal products].

Registration of Chinese patent medicine in European Union (EU) is of great significance to the internationalization of traditional Chinese medicine as EU market acts as an important position in the global botanical market. In retrospect, the domestic studies on EU regulations of traditional herbal medicinal products have been conducted for more than 10 years, but there is still some cognitive bias and lack of research. In this paper, a review of the relevant research progress and the main misunderstanding problems about Directive 2004/24/EC, like the centralized and decentralized supervision system of traditional herbal medicinal products in the EU, marketing authorization procedures for traditional herbal medicinal products, Community Herbal Monograph and List Entries, would be systematically analyzed, so as to provide reference for the registration of Chinese patent medicine in EU.

Validation of the G.LAB MD2680 digital automatic blood pressure monitor according to international protocols.

OBJECTIVE: The aim of this study was to validate the G.LAB MD2680 digital automatic blood pressure (BP) monitor according to major international protocols. PARTICIPANTS AND METHODS: The device was evaluated against auscultatory sphygmomanometry according to the European Society of Hypertension International Protocol (ESH-IP) revision 2010, the British Hypertension Society (BHS), and the International Organization for Standardization (ISO) 81060-2:2013 protocols. Bland-Altman plots were completed for systolic (SBP) and diastolic blood pressures (DBP), and the mean differences and SDs between the test device and the reference device were computed for all BP values. RESULTS: The G.LAB MD2680 passed the ESH-IP revision 2010 on 33 participants with a mean device-observer difference of 0.89±4.97 mmHg for SBP and 0.72±4.91 mmHg for DBP, respectively. The device achieved A/A grading for the BHS protocol among 85 participants with a device-observer difference of 0.70±6.35 mmHg for SBP and 0.62±6.41 mmHg for DBP, respectively. Furthermore, it also fulfilled the two criteria of the ISO 81060-2:2013 protocol. CONCLUSION: The G.LAB MD2680 digital automatic BP monitor fulfilled the accuracy requirements of the ESH-IP revision 2010 and the ISO 81060-2:2013 protocols, and achieved A/A grade of the BHS protocol, and it can be recommended for self-measurement in adults.

Regulation of pulmonary surfactant synthesis in fetal rat type II alveolar epithelial cells by microRNA-26a.

Pulmonary surfactant, a unique developmentally regulated, phospholipid-rich lipoprotein, is synthesized by the type II epithelial cells (AECII) of the pulmonary alveolus, where it is stored in organelles termed lamellar bodies. The synthesis of pulmonary surfactant is under multifactorial control and is regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, growth factors, estrogens, androgens, thyroid hormones, and catecholamines acting through beta-adrenergic receptors, and cAMP. While there is increasing evidence that microRNAs (miRNAs) are involved in the regulation of almost every cellular and physiological process, the potential role of miRNAs in the regulation of pulmonary surfactant synthesis remains unknown. miRNA-26a (miR-26a) has been predicted to target SMAD1, one of the bone morphogenetic protein (BMP) receptor downstream signaling proteins that plays a key role in differentiation of lung epithelial cells during lung development. In this study, we explored the regulation role of miR-26a in the synthesis of pulmonary surfactant. An adenoviral miR-26a overexpression vector was constructed and introduced into primary cultured fetal AECII. GFP fluorescence was observed to determinate the transfection efficiency and miR-26a levels were measured by RT-PCR. MTT was performed to analyze AECII viability. qRT-PCR and Western blotting were used to determine the mRNA and protein level of SMAD1 and surfactant-associated proteins. The results showed that miR-26a in fetal AECII was overexpressed after the transfection, and that the overexpression of miR-26a inhibited pulmonary surfactant synthesis in AECII. There was no significant change in cell proliferation. Our results further showed that overexpression of miR-26a reduced the SMAD1 expression both in mRNA and protein level in fetal AECII. These findings indicate that miR-26a regulates surfactant synthesis in fetal AECII through SMAD1.

[Bioinformatic analysis of target gene prediction and related signaling pathways of miRNA-126*].

OBJECTIVE: To bioinformatically predict and analyze target genes of miRNA-126(*), with the aim of providing certain basis for related research about target genes and regulatory mechanism in the future. METHODS: The miRNA chip technology was applied to measure expression levels of miRNA-126(*) in 3 time points (embryo 16, 19 and 21 days) of fetal lung development. Then the target genes of miRNA-126(*) were screened through miRGen2.0 database. Subsequent bioinformatic analysis of these target genes was performed by Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes Pathway analysis (KEGG Pathway analysis). RESULTS: miRNA-126(*) manifested continuously upregulated expression with the lung development (from embryo 16 to 21 days). There were 422 predicted target genes in total, and the gene set mainly located in glucuronosyltransferase activity, transferase activity (GO molecular function), multicellular organismal development, developmental process (GO biology process) and intracellular part (GO cellular component). The KEGG Pathway analysis demonstrated that the gene set mostly located in RNA degradation (signal transduction pathway) and prion diseases (disease pathway). CONCLUSIONS: The results suggest that miRNA-126(*) plays a certain role in fetal lung development and provide a basis for lung development research in the future.

[Role of miRNA-126/miRNA-126* in the fetal lung development of rats].

OBJECTIVE: To investigate the expression and role of miRNA-126/miRNA-126(*) in the fetal lung development of rats. METHODS: Twelve pregnant Sprague-Dawley rats were randomly divided into 3 groups and the fetal rats were removed at 16, 19 and 21 days of gestation respectively. Hematoxylin and eosin staining was performed to observe lung morphology of fetal rats. Then microRNA (miRNA) microarray was used to study the expression patterns of miRNA-126/miRNA-126(*) in fetal lungs at the three time points. And miRNA-126(*) was selected for further study by real-time PCR. RESULTS: There was no evident difference in the expression of miRNA-126 among the three groups, however the expression level of miRNA-126(*) increased gradually as the fetal lung developed. The real-time PCR result further showed that expression of miRNA-126(*) increased gradually with lung development, displaying significant differences among the three groups (P<0.05). CONCLUSIONS: miRNA-126(*) may play an important role in development of the fetal lung in rats.

Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells.

Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo. Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice. The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.

The Src homology 2 domain-containing adapter protein B (SHB) regulates mouse oocyte maturation.

SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb -/- mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/- and -/- mice revealed impaired early embryo development in the -/- embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development.

[Clinical study of glycated albumin measurement by enzymatic method in type 2 diabetes mellitus].

OBJECTIVE: To evaluate the clinical significance of glycated albumin (GA) measured by enzymatic method and to compare its effect with glycosylated hemoglobin A1c (HbA1c) in type 2 diabetes mellitus (DM). METHODS: 128 type 2 DM patients and 84 normal subjects from the Chinese PLA General Hospital were enrolled for the study. The levels of GA, HbA1c, FBG, PBG in DM patient were detected at baseline and followed visit at 2, 4, 8 weeks after blood glucose management. The levels of GA, HbA1c, FBG, PBG and 75 g OGTT were also detected in above normal subjects. RESULTS: Intra CV and inter CV of enzymatic were (0.74-0.9)% and (0.94-1.49)% respectively. In normal subjects GA was in the range of (9-14)%. At baseline, the GA level was significantly correlated with HbA1c (r = 0.8326, P < 0.01), FBG and 2 hour PBG. After 2, 4, 8 weeks treatment, GA level in DM patients was concomitantly decreased with the improvement of FBG, PBG and HbA1c. At early 2 weeks visit, GA, but not HbA1c, showed significant decrease from its baseline (P < 0.05). CONCLUSION: Enzymatic measuring GA was highly correlated with HbA1c, and changing concomitantly with the decrease of HbA1c, FBG, PBG during the 8 weeks treatment. GA was more sensitive than HbA1c for short-term variations of glycemic control during treatment of diabetic patients. GA can be used as a better index of short term mean level of blood glucose in diabetic patients.

[Characteristics of glycemic excursion in different subtypes of impaired glucose intolerance].

OBJECTIVE: To investigate the characteristics of glycemic excursion of different subtypes of glucose tolerance. METHODS: Assessed by oral glucose tolerance test (OGTT) repeated twice, 81 individuals were divided into 4 groups: normal glucose tolerance (NGT, n = 18), isolated impaired fasting glycemia (IFG, n = 12), isolated impaired glucose tolerance (IGT, n = 19), combined IFG/IGT (n = 11), and newly diagnosed type 2 diabetes mellitus (T2DM, n = 21). And then continuous glucose monitoring system (CGMS) was used for 72 hours to monitor the blood glucose (BG) level before drug intervention. RESULTS: (1) The levels of largest amplitude of glycemic excursions (LAGE), mean blood glucose (MBG), and standard deviation of mean level of blood glucose fluctuation (SDBG) increased gradually with the deterioration of glucose tolerance. The mean amplitude of glucose excursion (MAGE) readout of the IGT group was (3.2 +/- 1.2) mmol/L, significantly higher than that of the NGT group [(1.6 +/- 0.5) mmol/L, P < 0.05], and significantly lower than that of the T2DM group [(5.2 +/- 1.9) mmol/L, P < 0.05]. The level of frequency of glucose excursion (FGE) decreased along with the decrease of glucose tolerance: NGT group [(6.1 +/- 3.4)] > IGT/IFG group [(5.5 +/- 2.5)] > T2DM group [(4.8 +/- 1.8)]. Among the three components of IGR, the IGT group showed highest MAGE (3.2 +/- 1.2) mmol/L and lowest FGE level (4.9 +/- 1.8). (2) The level of absolute mean of daily difference (MODD) increased in the following order: NGT group [(0.8 +/- 0.3) mmol/L], IGT group [(1.1 +/- 0.4) mmol/L], IFG/IGT group [(1.2 +/- 0.4) mmol/L], and T2DM group [(2.0 +/- 1.0) mmol/L] (all P < 0.05). (3) The fasting glucose level deteriorated the most rapidly in the IFG group, while it reached the highest postprandial peak in the IFG/IGT group. The blood glucose curve increased along the order of NGT, I-IGT, IFG/IGT, IFG, and T2DM. (4) When the level of glycosylated hemoglobin (HbA1c) level was less than 7%, the fasting phase of curve virtually coincided with each other among individual groups with different HbA1c levels; however, the postprandial peak separated slightly. When the HbA1C level was between 7.0% and 7.9%, the postprandial peaks of individual groups with different HbA1c levels dispersed markedly. When the HbA1c level was higher than 8%, the fasting blood glucose curve moved upwards significantly with increasing postprandial excursion. CONCLUSION: (1) With the deterioration of glucose regulation, the intraday and day-to-day blood glucose excursions become increasingly fluctuant. (2) The amplitude of glycemic excursion is lower in the NGT group than in the T2DM group, however, the frequency of glycemic excursion is higher in the NGT subject than in the T2DM subjects. The glucose excursion profile of the IGR subjects is between the NGR and T2DM subjects. (3) The characteristics of glucose excursion of the IGT group are similar to those of the T2DM group, and the characteristics of the IFG group are similar to those of the NGT group. (4) The loss of postprandial glycemic control precedes evident deterioration in fasting phase of IGR.

Shb null allele is inherited with a transmission ratio distortion and causes reduced viability in utero.

SHB is an Src homology 2 domain-containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb-/- pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb- gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/-) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb-/- animals, but increased number of Shb+/- animals. The Shb- allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb-/- offspring. Shb-/- animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/- female mice ovulated preferentially Shb- oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development.

An activating mutation in the PDGF receptor-beta causes abnormal morphology in the mouse placenta.

An oncogenic D842V mutation in the platelet-derived growth factor (PDGF) alpha-receptor (Pdgfra) has recently been described in patients with gastrointestinal stromal tumors. In order to test if the same mutation would confer oncogenic properties to the homologous PDGF beta-receptor (Pdgfrb), the corresponding aspartic acid residue at position 849 of Pdgfrb was changed into valine (D849V) using a knock-in strategy. This mutation turned out to be dominantly lethal and caused death even in chimeras (from 345 transferred chimeric blastocysts, no living coat chimeras were detected). Experiments employing mouse embryonic fibroblasts (MEFs) indicated hyperactivity of the mutant receptor. The mutant receptor was phosphorylated in a ligand-independent manner and, in contrast to wild-type MEFs, mutant cells proliferated even in the absence of ligand. Knockout experiments have previously indicated a role for Pdgfrb in placental development. We therefore analyzed wild-type and Pdgfrb D849V chimeric placentas from different gestational stages. No differences were detected at embryonic days 11.5 and 13.5 (n=4). At embryonic day 17.5, however, chimeric placentas (n=3/4) displayed abnormalities both in the labyrinth and in the chorionic plate. The changes included hyper-proliferation of alpha-smooth muscle actin and platelet/endothelial cell adhesion molecule-1 positive cells in the labyrinth and cells in the chorionic plate. In addition, the fetal blood vessel compartment of the labyrinth was completely disorganized.

The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice.

The Arctic mutation (APP E693G) is unique, since it is located within the amyloid-beta (Abeta) sequence and leads to Alzheimer's disease (AD). Arctic Abeta peptides more easily form Abeta protofibrils in vitro, but little is known about the pathogenic mechanism of the Arctic mutation in vivo. Here, we analyzed APP transgenic mice with both the Swedish and Arctic mutations (tg-APPArcSwe) and transgenic mice with the Swedish mutation alone (tg-APPSwe). Intense intraneuronal Abeta-immunoreactive staining was present in young tg-APPArcSwe mice, but not in tg-APPSwe mice. Intracellular Abeta aggregates in tg-APPArcSwe were strongly stained by antibodies recognizing the N-terminus of Abeta, while those recognizing the C-terminus of Abeta stained weakly. The Abeta aggregates inside neurons increased with age and predated extracellular Abeta deposition in both tg-APPArcSwe and tg-APPSwe mice. Senile plaque deposition was markedly accelerated in tg-APPArcSwe mice, as compared to tg-APPSwe mice. We conclude that the Arctic mutation causes AD by facilitating amyloidosis through early accumulation of intracellular Abeta aggregates in association with a rapid onset of senile plaque deposition.

Gli1 is not required for Pdgfralpha expression during mouse embryonic development.

Pdgfra is expressed in the mesenchyme of multiple organs during embryonic development and Pdgfralpha is involved in cell proliferation, differentiation, migration, and apoptosis in many tissues. A fine-tuned regulation of gene transcription is required to achieve these effects. To investigate if the Shh signaling pathway is involved in the tightly regulated Pdgfra expression during embryogenesis, we systematically compared Gli1 and Pdgfralpha mRNA expression patterns in vivo from mouse embryonic day 9.5 to 14.5. We found that an initial partly overlapping expression of Gli1 and Pdgfralpha in the mesenchyme of foregut and somites was changed to different expression patterns when the mesenchyme differentiated into specialized structures such as intestinal villi and chondrocytes. Gli1 and Pdgfra were also expressed differently in the developing lung, heart, central nervous system, skin, tooth, and eye. Importantly, neither Pdgfralpha mRNA patterns nor levels were altered in Ihh mutant embryos although Gli1 and Ptc mRNA levels were dramatically reduced. Our results demonstrate that Gli1 is not required to induce Pdgfra expression during embryonic bone development, and are consistent with previous findings that Pdgfralpha and Hh pathways serve different functions in, e.g., bone, gut, and lung development. However, we cannot exclude the possibility that Glis can have more complex regulatory effects on Pdgfra gene activity, nor can we exclude such effects in pathological conditions.

p53 must be competent for transcriptional regulation to suppress tumor formation.

In vitro studies suggest that effective tumor suppression by p53 requires multiple domains to execute transcription-dependent and transcription-independent functions. We generated a mutant p53 allele in mice, p53(W25QL26S) (p53(QS)), containing an inactive transactivation domain to evaluate the importance of transactivation for p53-mediated tumor suppression. Recently, we discovered that the allele also contains a valine substitution for alanine at codon 135, which borders the DNA-binding domain. We found that p53(QSval135) bound to chromatin albeit less well than p53(QSala135), but both were equally deficient in transcriptional regulation, apoptosis induction in mouse embryo fibroblasts (MEFs), and suppression of tumor formation by E1A, Ha-Ras transformed MEFs. p53(QSval135) mice and p53-null mice exhibited identical tumor development kinetics and spectra in spontaneous and oncogene-initiated tumorigenicity assays, when tested in a homo- and heterozygous configuration. The p53(QSval135) allele did not have dominant negative functions and behaved as a null allele. Taken together, these data indicate that effective tumor suppression requires the transcriptional regulation function of p53, and they suggest that transactivation independent functions of p53 are unlikely to contribute significantly to tumor suppression in vivo.

Forced expression of platelet-derived growth factor B in the mouse cerebellar primordium changes cell migration during midline fusion and causes cerebellar ectopia.

The platelet-derived growth factor (PDGF) and receptors are expressed in the developing central nervous system and in brain tumors. To investigate the role of PDGF during normal cerebellar development, we created transgenic mice where PDGF-B was introduced into the endogenous Engrailed1 locus (En1). These mice expressed PDGF-B in all types of cells that constitute the developing cerebellum, with localized high expression in the ventral midline of the cerebellar anlage. This affected cell migration in the midline during fusion of the cerebellar anlage and caused misplacement of midline structures. PDGFR-alpha- and laminin alpha1-positive meningeal cells migrated inwards, attracted by the ectopic transgene expression in the ventral neuroepithelium. Other cells followed the meningeal cells and in the adult mouse, cells from all cortical cell layers were found misplaced in the midline. Moreover, the transgene caused an enhancement of capillary vessels. The findings indicate that normal PDGF signaling is important for proper neural tube fusion. It also illustrates that meningeal structures can influence the process.

Oligodendrocyte precursor hypercellularity and abnormal retina development in mice overexpressing PDGF-B in myelinating tracts.

Platelet-derived growth factor (PDGF) influences the generation of neurons and glia during embryogenesis and in early postnatal life. In an attempt to determine the consequences of an overexpression of PDGF-B during the first weeks of life, we targeted transgenic expression of a human PDGF-B cDNA to myelinating tracts using the promoter region of the myelin basic protein (MBP) gene. Transgenic mRNA and protein were expressed in the brain and the expression profile of the human PDGF-B during early postnatal development closely paralleled that of the endogenous mouse MBP gene. The gross morphological appearance of transgenic brains was normal but at the cellular level several phenotypic alterations could be identified. In white matter tracts such as the corpus callosum and cerebellar medulla, there was a marked hypercellularity. The number of oligodendrocyte precursors was increased and astrocytes were more abundant. In adult mice carrying the MBP-PDGF-B transgene, however, myelination appeared normal and the amount of oligodendrocytes was similar to that of control littermates. In addition to the phenotypic alterations in the brain, investigation of eye structure revealed a striking disorganization of retinal architecture. The retina was folded with cells collected in papillar or follicular-like structures. Retinal whole mount preparations after India ink perfusion revealed capillary disorganization with large-caliber vessels supporting only a few fine branches. Our observations strengthen the notion that PDGF is an important effector molecule in postnatal CNS development.