恒河猴心房利钠多肽的分离、纯化及活性研究

<正> 1983年以来,人们相继从人和大鼠等动物的心房中分离到心房利钠多肽(ANP)。但对非人灵长类动物——恒河猴的ANP了解甚少。由于ANP对机体循环系统的调节起着重要作用,其强大的降压、利钠、利尿的生理功能,在心血管疾病的防治方面具有重要意义。我们曾报导过恒河猴心房肌细胞中存在着大量的ANP样物质,随后对此物质进行了分离、纯化及活性检测,现简报如下。     

Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Background

Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration.

Methods

Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested.

Findings

LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages.

Conclusion

Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.     

葡萄病毒B的RT-PCR检测技术研究

旨在建立适合葡萄的葡萄病毒B的RT-PCR检测技术。从感染葡萄病毒B的葡萄皮层中提取总RNA,以其为模板合成cDNA第一链,并利用两对引物分别进行PCR扩增;回收PCR特异扩增产物,与pUCm-T载体连接,并进行转化、重组克隆的筛选、重组质粒的酶切鉴定和序列测定。结果显示,两对引物均能获得与预期片段大小一致长约460 bp和470 bp的扩增产物扩增片段序列与已报道GVB序列(序列号:X75448)的核苷酸同源性均为81%,经反复多次试验证实,利用总RNA为模板合成cDNA并进行PCR扩增检测GVB是准确、可靠的。     

Neurovascular musculus obliquus internus abdominis flap free transfer for facial reanimation in a single stage

A study of the anatomy and transplantation of the musculus obliquus internus abdominis with a neurovascular pedicle transfer for facial reanimation in one stage is presented. Eleven adult cadavers (22 face sides) were dissected to observe the shape, thickness, innervation, and blood supply of the musculus obliquus internus abdominis. The blood supply of this muscle primarily comes from the musculus obliquus internus abdominis branch of the deep circumflex iliac artery (diameter, 1.3 +/- 0.2 mm), but it can also come from the eleventh intercostal artery (diameter, 1.14 +/- 0.3 mm) and the infracostal artery (diameter, 1.5 +/- 0.2 mm). The branch of the deep circumflex iliac artery and its vena comitans, or the infracostal artery and its vena comitans, could be anastomosed for muscle transplantation. The innervation of the musculus obliquus internus abdominis comes from the tenth and eleventh intercostal nerves (length, 12.7 +/- 1.5 cm) and the infracostal nerve (length, 12.9 +/- 1.3 cm). The eleventh intercostal nerve and the infracostal nerve were selected for anastomosis of muscle transplantation. From November of 1995 to November of 1999, 14 patients with long established facial paralysis were treated with transplantation of a musculus obliquus internus abdominis flap in one stage and were followed for 10 months to 6 years. In 13 patients, the dynamic functions of the transplanted muscles were restored, the obliqueness of the mouth and philtrum while static was corrected, and the facial muscle activities while smiling were harmonized. The eyelids of the paralyzed side could be closed postoperatively, indicating that the function of the orbicularis oculi of the paralyzed side was restored. The single-stage transplantation of a free musculus obliquus internus abdominis flap with one vascular, multi-nerve pedicle is a new method for facial reanimation in the treatment of long established facial paralysis. Because of the simplicity of the procedure and the completeness of the functional reanimation of the paralyzed facial muscles, compared with the results of other free muscle flap transfers, it is an ideal procedure for facial reanimation.     

Cognitive Changes during Prolonged Stay at High Altitude and Its Correlation with C-Reactive Protein

Hypersensitive C-reaction protein (hsCRP) may be a risk factor for cognitive impairment resulting from Alzheimer’s disease (AD), stroke, and vascular dementia. This study explored the correlation of peripheral blood hsCRP level with cognitive decline due to high altitude exposure. The study was conducted on 100 male military participants who had never been to high altitude. Cerebral oxygen saturation monitoring, event related potentials (P300, N200) detection, and neurocognitive assessment was performed and total hsCRP, interleukin-6 (IL-6), and homocysteine was estimated at 500m altitude, 3650m altitude, 3day, 1, and 3 month post arriving at the base camp (4400m), and 1 month after coming back to the 500m altitude. High altitude increased brain oxygen saturation, prolonged P300 and N200 latencies, injured cognitive functions, and raised plasma hsCRP levels. But they all recovered in varying degrees at 1 and 3 month post arriving at the base camp (4400m). P300 latencies and hsCRP levels were strongly correlated to cognitive performances. These results suggested that cognitive deterioration occurred during the acute period of exposure to high altitude and may recover probably owning to acclimatization after extended stay at high altitude. Plasma hsCRP is inversely correlated to neurological cognition and it may be a potential biomarker for the prediction of high altitude induced cognitive dysfunction.     

ALPK2 acts as tumor promotor in development of bladder cancer through targeting DEPDC1A

Bladder cancer is one of the most common malignant tumors in the urinary system. The development and improvement of treatment efficiency require the deepening of the understanding of its molecular mechanism. This study investigated the role of ALPK2, which is rarely studied in malignant tumors, in the development of bladder cancer. Our results showed the upregulation of ALPK2 in bladder cancer, and data mining of TCGA database showed the association between ALPK2 and pathological parameters of patients with bladder cancer. In vitro and in vivo experiments demonstrated that knockdown of ALPK2 could inhibit bladder cancer development through regulating cell proliferation, cell apoptosis, and cell migration. Additionally, DEPDC1A is identified as a potential downstream of ALPK2 with direct interaction, whose overexpression/downregulation can inhibit/promote the malignant behavioral of bladder cancer cells. Moreover, the overexpression of DEPDC1A can rescue the inhibitory effects of ALPK2 knockdown on bladder cancer. In conclusion, ALPK2 exerts a cancer-promoting role in the development of bladder cancer by regulating DEPDC1A, which may become a promising target to improve the treatment strategy of bladder cancer.Subject terms: Cancer models, Bladder cancer     

Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic β cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 β cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in β cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor–PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of β cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.Glucagon-like peptide-1 (GLP-1)¹ is a gastrointestinal hormone secreted by intestinal L cells upon food intake that is best known for its role in controlling glucose homeostasis. Acting through its cognate glucagon-like peptide-1 receptor (GLP-1R), GLP-1 has several important physiological and pharmacological functions. GLP-1 is best known for enhancing glucose-stimulated insulin secretion (GSIS) from the pancreatic β cells. Importantly, the insulinotropic properties of GLP-1 are maintained in patients with type 2 diabetes (1), which is characterized by insufficient insulin secretion from pancreatic β cells and an inability to maintain glucose homeostasis. Therefore, therapeutic strategies targeting GLP-1R have been developed to treat type 2 diabetes (2, 3). In addition to augmenting insulin secretion, GLP-1 has been known to improve glucose sensing, proinsulin biosynthesis, survival, and proliferation of β cells (3, 4) in a variety of experimental models. GLP-1 also has several extrapancreatic effects, including actions on the central nervous system to inhibit food intake (5), the stomach to decrease gastric emptying and gastric acid secretion (6), and the lungs to stimulate secretion of macromolecules from airways (7). Additionally, GLP-1 has an effect on the heart and possibly the kidney to modulate blood pressure and heart rate (8, 9).The GLP-1R is a member of the B1 family of G protein–coupled receptors (secretin receptor family). In mammals, GLP-1R is expressed in multiple tissues, including pancreatic β cells and δ cells (10), hypothalamus, lung, stomach, heart, kidney (11), and thyroid (12), which in part explains its diverse actions. Upon ligand binding, the GLP-1R is capable of coupling to diverse cell signal transduction pathways, but it is best known for its actions on G protein Gs α and adenylate cyclase activity to increase intracellular cAMP. It is known that other proteins can affect GLP-1R activity in addition to G proteins, including β-arrestin and caveolin, which affect receptor internalization and trafficking. β-Arrestin 1 is also required for proper GLP-1-stimulated cAMP production (13–15). More recently, it was shown that another B1 family member, gastric inhibitory polypeptide receptor heterodimerizes with GLP-1R, decreasing GLP-1-induced β-arrestin recruitment and mobilization (16). Very recently, our group identified several novel potential GLP-1R interactors using a membrane-based split-ubiquitin yeast two-hybrid (MYTH) assay (17). Three β cell–expressing membrane-bound interactors, solute carrier family 15 member 4 (SLC15A4), amyloid β A4 precursor-like protein 1 (APLP1), and adaptor-related protein complex 2 subunit mu (AP2M1), were further selected for individual knockdown in mouse insulinoma (MIN6) β cells using small interfering RNAs (siRNAs). GLP-1-induced insulin secretion was significantly enhanced when these genes were silenced, suggesting that these interactor proteins attenuate GLP-1R activity. These findings demonstrated that GLP-1R protein interactions are complex and the interactors can have measurable effects on receptor trafficking and downstream signaling. Such interactions may in part explain the diverse tissue-specific effects of GLP-1 and offer avenues for controlling GLP-1 actions in a tissue-selective manner.Although the MYTH system is well established (18) and has been applied to study G protein–coupled receptor interactomes (17), it is limited on two fronts. Firstly, it must be performed in yeast which is not an ideal representation of the mammalian system. Secondly, it is technically difficult to activate the receptor in MYTH, thus, effects of ligand stimulation on the receptor interactome cannot be assessed. Recently, affinity purification–mass spectrometry (AP-MS) has become a powerful tool for discovering and examining novel protein–protein interactions, including those between membrane-bound proteins in mammalian cells (19–21). In the current study, we applied AP-MS to discover novel GLP-1R interactors and employed a human GLP-1R harboring a FLAG^® epitope. GLP-1R-Flag was expressed in either Chinese hamster ovary (CHO) cells or MIN6 β cells, and interactors were studied in the presence or absence of GLP-1.     

ERK-mediated Cytoplasmic Retention of USP11 Contributes to Breast Cancer Cell Proliferation by Stabilizing Cytoplasmic p21

Breast cancer ranks as the most frequently diagnosed cancer among women worldwide. Elevated cytoplasmic p21 levels are often found in breast cancer tissues and related to a poor prognosis. However, the underlying mechanisms that lead to the stabilization of cytoplasmic p21 protein, which normally has a very short half-life, remain obscure. In this study, we found that there was a strong correlation between p21 and USP11 in the cytoplasm of breast cancer tissues and cells. Furthermore, we revealed that ERK1/2 phosphorylated USP11 at the Ser905 site, which promoted the cytoplasmic localization of USP11. In the cytoplasm, USP11 colocalized and interacted with p21. As a result, USP11 catalyzed the removal of polyubiquitin chains bound to cytoplasmic p21 and resulted in its stabilization. Functionally, USP11-mediated stabilization of cytoplasmic p21 induced breast cancer cell proliferation in vitro and in vivo. Our findings provide the first evidence that ubiquitinated p21 in the cytoplasm can be recycled through USP11-mediated deubiquitination, and we identified the USP11-p21 axis in the cytoplasm as a potential therapeutic target for breast cancer control.     

Periplocymarin alleviates pathological cardiac hypertrophy via inhibiting the JAK2/STAT3 signalling pathway

Pathological cardiac hypertrophy is the most important risk factor for developing chronic heart failure. Therefore, the discovery of novel agents for treating pathological cardiac hypertrophy remains urgent. In the present study, we examined the therapeutic effect and mechanism of periplocymarin (PM)‐mediated protection against pathological cardiac hypertrophy using angiotensinII (AngII)‐stimulated cardiac hypertrophy in H9c2 cells and transverse aortic constriction (TAC)‐induced cardiac hypertrophy in mice. In vitro, PM treatment significantly reduced the surface area of H9c2 cells and expressions of hypertrophy‐related proteins. Meanwhile, PM markedly down‐regulated AngII‐induced translocation of p‐STAT3 into the nuclei and enhanced the phosphorylation levels of JAK2 and STAT3 proteins. The STAT3 specific inhibitor S3I‐201 or siRNA‐mediated depleted expression could alleviate AngII‐induced cardiac hypertrophy in H9c2 cells following PM treatment; however, PM failed to reduce the expressions of hypertrophy‐related proteins and phosphorylated STAT3 in STAT3‐overexpressing cells, indicating that PM protected against AngII‐induced cardiac hypertrophy by modulating STAT3 signalling. In vivo, PM reversed TAC‐induced cardiac hypertrophy, as determined by down‐regulating ratios of heart weight to body weight (HW/BW), heart weight to tibial length (HW/TL) and expressions of hypertrophy‐related proteins accompanied by the inhibition of the JAK2/STAT3 pathway. These results revealed that PM could effectively protect the cardiac structure and function in experimental models of pathological cardiac hypertrophy by inhibiting the JAK2/STAT3 signalling pathway. PM is expected to be a potential lead compound of the novel agents for treating pathological cardiac hypertrophy.     

叉蕨科30种植物叶表皮形态特征研究

利用光镜对叉蕨科7属30种植物叶表皮形态特征进行详细观察研究。结果显示：（1）叉蕨科30种植物的叶上表皮和下表皮细胞形状均为不规则型,垂周壁式样为深波状或浅波状,具单晶或针晶;上表皮细胞的长宽比为1.62~4.0,下表皮细胞的长宽比为1.63~3.06。（2）在30种植物中共观察到7种气孔器类型,分别为：极细胞型、腋下细胞型、聚合极细胞型、聚腋下细胞型、不等细胞型、无规则四细胞型和不规则型,每种植物分别具有4~7种气孔器类型,均为下生型气孔;气孔长宽比为1.22~1.91,气孔密度为8~76个/mm²,气孔指数为3.9%~25.7%。（3）基于气孔器类型组成进行聚类分析,可将30种植物分成3个类群。（4）对叶表皮形态特征分析认为,轴脉蕨属应介于叉蕨属和肋毛蕨属之间,且与叉蕨属关系更近;叉蕨属的范畴还有待进一步研究;支持将肋毛蕨属从叉蕨科中分离出来置于鳞毛蕨科,但不支持黄腺羽蕨属归入鳞毛蕨科。