抗生素胞内抗菌活性及其细胞药代动力学评价的研究进展

侵入宿主细胞内的病原菌不仅逃避了体液免疫,而且逃避了抗生素的作用;同时抗生素在胞内的抗菌活性往往很差,这也是一些感染性疾病反复发作、迁延不愈的主要原因之一。现有的基于血浆药物浓度的经典药代动力学研究不足以有效地预测体内抗生素药效,因此迫切需要开展抗生素的细胞药代动力学研究。本文就近年来抗生素细胞药代动力学及胞内抗菌活性的相关研究做一综述,总结影响抗生素胞内活性的PK/PD参数,并且讨论优化其胞内活性的策略。

腺相关病毒载体介导人组织激肽释放酶结合蛋白的胞内生物活性研究

人组织激肽释放酶结合蛋白(Kallistatin,KAL)是一种分泌型蛋白,其通过与胞外受体结合来调控多条下游信号通路,但尚未有KAL的胞内活性研究以及胞内KAL是否具有与胞外相似生物学活性的报道。通过构建无信号肽的KAL腺相关病毒表达载体(rAAV-NSK)来探索KAL蛋白的胞内活性。转染rAAV-NSK后,所有受试细胞均有KAL胞内表达但不分泌,其中HUVEC的增殖、迁移和小管形成均受到抑制;NCI-H460、NCI-H446和A549受到不同程度抑制。这一细胞水平研究,不仅证实了KAL的胞内活性,也暗示着KAL将可能作为一种"平衡因子"参与多靶点调控,也为解释目前有关KAL在PI3K-Akt信号通路的调控矛盾提供了新的思路。

Effect of Platelet Activation and Endothelial Cell Injury on Malignant Cancer

Objective: To study the effects of platelet activation and endothelial cell injury on the patients with malignant tumor and their prognoses.Methods: Radioimmunity and ELISA methods were employed to detect the TXB2, GMP-140, vWF, cGMP and FN in 78 cases of malignant tumor and 40 healthy control persons.Results: The levels of TXB2, MP-140 and cGMP were increased in intestinal cancer group, lung cancer group and hepatic cancer group, while FN decreased in intestinal cancer and lung cancer group. cGMP was positively related to TXB2, GMP-140, vWF in malignant tumor group. FN was decreased in the group complicated with infection and the group with metastasis, while the other indexes increased. GMP-140, vWF and cGMP was decreased after operation except for the increasing of FN.Conclusion: Activations of platelet and injury of endothelial cells developed in patients with malignant tumor, and both of them affected the metastasis and prognosis of malignant tumor. Key words platelet activation - epithelium injury - malignant tumor - metastasis This work was supported by grants from Guangdong Medical Science foundation (A2000633).

Optimization for Production of Intracellular Polysaccharide from Cordyceps ophioglossoides L2 in Submerged Culture and Its Antioxidant Activities in vitro

Cordyceps ophioglossoides is a valuable traditional medicinal material.We have found that intracellular polysaccharide(IPS) is the major biologically active ingredient in Cordyceps ophioglossoides.This study is the first time to optimize the yield of IPS from Cordyceps ophioglossoides.The optimal medium for IPS production consists of glucose 54.50 g·L·1,yeast powder 25.50 g·L·1,NaH2PO4 0.4 g·L·1 and K2HPO4 0.4 g·L·1.The suggested culture conditions are 24 ℃,initial pH 4.5 with a rotary speed of 120 r·min·1 for 168 h.The yield of IPS is 737.93 mg·L·1,which is 50% higher than the yield under the conditions prior to optimization.The anti-oxidative activities of IPS in Cordyceps ophioglossoides L2 are also characterized using various in vitro assay.The anti-oxidative activity may explain the reason why IPS from Cordyceps ophioglossoides can be used to fight against neurodegenerative dis-eases and menopausal symptoms.

Alphastatin downregulates vascular endothelial cells sphingosine kinase activity and suppresses tumor growth in nude mice bearing human gastric cancer xenografts

AIM： To investigate whether alphastatin could inhibit human gastric cancer growth and furthermore whether sphingosine kinase （SPK） activity is involved in this process. METHODS： Using migration assay, MTT assay and Matrigel assay, the effect of alphastatin on vascular endothelial cells （ECs） was evaluated in vitro. SPK and endothelial differentiation gene （EDG）-1, -3, -5 mRNAs were detected by reverse transcription-polymerase chain reaction （RT-PCR）. SPK activity assay was used to evaluate the effect of alphastatin on ECs. Matrigel plug assay in nude mice was used to investigate the effect of alphastatin on angiogenesis in vivo. Female nude mice were subcutaneously implanted with human gastric cancer cells （BGC823） for the tumor xenografts studies. Micro vessel density was analyzed in Factor Ⅷ-stained tumor sections by the immunohistochemical SP method. RESULTS： In vitro, alphastatin inhibited the migration and tube formation of ECs, but had no effect on proliferation of ECs. RT-PCR analysis demonstrated that ECs expressed SPK and EDG-1, -3, -5 mRNAs. In vivo, alphastatin sufficiently suppressed neovascularization of the tumor in the nude mice. Daily administration of alphastatin produced significant tumor growth suppression. Immunohistochemical studies of tumor tissues revealed decreased micro vessel density in alphastatin-treated animals as compared with controls. CONCLUSION： Downregulating ECs SPK activity may be one of the mechanisms that alphastatin inhibits gastric cancer angiogenesis. Alphastatin might be a useful and relatively nontoxic adjuvant therapy in the treatment of gastric cancer.

How to protect liver graft with nitric oxide

Organ preservation and ischemia reperfusion injury associated with liver transplantation play an important role in the induction of graft injury. One of the earliest events associated with the reperfusion injury is endothelial cell dysfunction. It is generally accepted that endothelial nitric oxide synthase （e-NOS） is cell-pro- tective by mediating vasodilatation, whereas inducible nitric oxide synthase mediates liver graft injury after transplantation. We conducted a critical review of the literature evaluating the potential applications of regulating and promoting e-NOS activity in liver preservation and transplantation, showing the most current evidence to support the concept that enhanced bioavailability of NO derived from e-NOS is detrimental to ameliorate graft liver preservation, as well as preventing subse- quent graft reperfusion injury. This review deals mainly with the beneficial effects of promoting ＂endogenous＂ pathways for NO generation, via e-NOS inducer drugs in cold preservation solution, surgical strategies such as ischemic preconditioning, and alternative ＂exogenous＂ pathways that focus on the enrichment of cold storage liquid with NO donors. Finally, we also provide a basic bench-to-bed side summary of the liver physiology and cell signalling mechanisms that account for explaining the e-NOS protective effects in liver preservation and transplantation.

Expression and proteolytic activity of calpain in lens epithelial cells of oxidative cataract

Objective: To study the role of calpain in the mechanism of oxidative cataract through detecting the level of intracellular free Ca2+, the expression and proteolytic activity of calpain in the lens epithelial cells (LECs) of H2O2-induced cataract. Methods: Rat lenses were cultured in vitro and cataract was induced by H2O2. The level of intracellular free Ca2+ was measured by fluorescence determination with fura-2/AM. The expression of m-calpain protein in LECs was detected with immunohistochemical method. The proteolytic activity in LECs was measured using a fluorogenic synthetic substrate. Results: There were significant differences of the level of intracellular free Ca2+ (P=0.001, 0.000, 0.000), the expression of m-calpain (P=0.001, 0.000, 0.000) and the proteolytic activity of calpain (P=0.001, 0.000, 0.000) between H2O2-induced and control group at 6, 12 and 24 h, respectively. Conclusions: H2O2 can increase intracellular free Ca2+, then enhance the expression and proteolytic activity of calpain which may play a role in the mechanism of oxidative cataract of rat.

Gradient decrease in telomerase and its RNA in progressive passage of human endometrial stromal stem cells

The purpose of this study was to explore the change of telomerase in passage from human endometrial stromal stem cells isolated from human endometrium.Telomerase activity of cultured endometrial stromal cells was assessed at mRNA and protein levels using RT-PCR and immunohistochemistry technique.Telomerase mRNA and protein levels were higher at early passages,and then had a gradually decreased trend of immunoreactive intensity and gradually weakened positive cells with progressive passage in endometrial stromal stem cells.These results suggest that telomerase is contributed to the insenecence of endometrial stem cell.

hVEGF165 Expression in Escherichia coli Conserves Its Biological Function

The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor 2, located on endothelial cells lining the surface of blood vessels. This binding stimulates the cascade of downstream signalling events leading to process known as angiogenesis, hVEGF165 overexpressed with His-tag in BL21 E. coli cells forms inclusion bodies （insoluble protein）, so the research found the procedure for its solubilization and purification on a Nickel based affinity chromatography. Although this eukaryotic signal protein needs posttranslational processing for its full function as a homodimer, author verified the biological activity of our hVEGF165 protein, obtained as monomer, by wound healing test.

Recent advances in cell imaging and cytotoxicity of intracellular stimuli-responsive nanomaterials

The stimuli-responsive nanomaterials are gaining more and more interest in the biological field,including cell imaging and biosensing etc. Nanomaterials in response to the bio-relevant stimuli(i.e., p H, enzymes and other bioactive molecules) can be utilized to enhance imaging(i.e., optical imaging, MRI, and multi-mode imaging) sensitivity via disease site-specific delivery and controlled release, which helps to diagnose cancer at an early stage or to monitor progression during treatment. In the triggered responsive process, smart nanomaterials undergo changes in physiochemical properties that can cause cytotoxicity or influence on cell functions due to the interactions between nanomaterials and cells. In order to promote the design and fabrication of effective platforms for therapeutics and diagnostics, special attention should be paid to these effects. By taking the advantages of intracellular stimuli, the controlled self-assembly in living cells can be achieved, which has been used for various in situ detections and insights into biological self-assembly. In this review, the recent advances in cell imaging, cytotoxicity and self-assembly of intracellular stimuli-responsive nanomaterials are summarized. Some principles for the further design and applications of intracellular stimuli-responsive nanomaterials and future perspectives are discussed.