重组人粒细胞巨噬细胞集落刺激因子的生物质谱和毛细管电泳法

目的：为准确分析重组人粒细胞巨噬细胞集落刺激因子(rHuGM-CSF)半成品及其制剂的纯度和表达的正确性。方法：用毛细管区带电泳测定了2批rHuGM-CSF半成品和3批rHuGM-CSF注射液的纯度。用基体辅助激光解吸飞行时间质谱和电喷雾质谱测定了2批rHuGM-CSF半成品的分子量。用毛细管等电聚集法测定了rHuGM-CSF半成品的等电点。结果：2批半成品中1批含有少量二聚体,且含有4个不同等电点的组分,另1批表达不正确;2批成品均存在大量杂质。结论：发现有些rHuGM-CSF样品有生物学活性,但表达不正确,纯度也不高;各分析方法是互补的,不能相互替代。     

重组人粒细胞-巨噬细胞集落刺激因子治疗胃溃疡

哈药集团技术中心 1 前言 重组人粒细胞-巨噬细胞集落刺激因子(英文名称:Recombinant human granulocyte-macrophage colony stimulating factor,简称rhGM-CSF)作为九十年代以来高新技术生物工程产品,具有促进骨髓粒系造血的恢复,升高外周血白细胞数的作用,现已广泛应用于白血病及肿瘤病人的大剂量放化疗,严重感染等白细胞减少性疾病的治疗.     

重组人粒细胞巨噬细胞集落刺激因子比色测定中最适波长的研究

目的：研究重组人粒细胞巨噬细胞集落刺激因子比色测定中的最适波长。方法：采用UV－2001紫外分光光度计在0－700nm波长处测定其吸收度，绘制标准曲线。结果：该法测定样品在595nm处有最大吸收度，在695处无吸收。结论：重组人粒细胞巨噬细胞集落刺激因子比色测定中的最适波长为595nm。     

大肠杆菌表达重组人粒细胞—巨噬细胞集落刺激因子的中试纯化

重组人粒细胞 -巨噬细胞集落刺激因子 (rHuGM -CSF)表达产物在大肠杆菌中以包涵体的形式存在。方法　包涵体高压匀浆破菌抽提后 ,经凝胶过滤层析、复性、离子交换层析及凝胶过滤层析等纯化步骤。结果终产物纯度达 97% ,比活性达 1 2× 10 7U/mg蛋白 ,测定N端 2 0个氨基酸系列与其DNA系列推导的氨基酸系列完全一致。     

重组人粒细胞-巨噬细胞集落刺激因子治疗早期糖尿病足溃疡疗效观察

目的:观察局部皮下注射重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)治疗糖尿病足溃疡患者的疗效。方法:36例糖尿病足溃疡1～3级患者均用胰岛素强化控制血糖在理想范围内,溃疡面先用强力碘清洁消毒处理,再用生理盐水冲洗,清除坏死组织,第2次用生理盐水冲洗,然后随机分为2组。治疗组:直接将rhGM-CSF注射剂按5μg.kg-1.d-1沿创面周围皮下注射,每日1次;对照组:常规消毒清洁创面后,用无菌凡士林纱布覆盖溃疡面,每天换药1次,2组均治疗30d。结果:治疗组与对照组的总有效率分别为100.0%、83.3%(P<0.05);平均住院时间分别为21、32d(P<0.05)。结论:rhGM-CSF局部皮下注射较常规换药可提高糖尿病足溃疡1～3级患者的总有效率,促进糖尿病足慢性创面的愈合。     

重组人粒细胞巨噬细胞集落刺激因子凝胶剂治疗溃疡药效学研究

目的观察重组人粒细胞巨噬细胞集落刺激因子(gm-csf)凝胶剂治疗溃疡的药效。方法在豚鼠背部造溃疡模型,将凝胶剂外涂于豚鼠背部的溃疡处,不同时间内考察gm-csf凝胶剂对溃疡的药效。结果gm-csf凝胶剂组及gm-csf原液组同盐水组、基质组比较,溃疡面积明显缩小,有显著差异。结论gm-csf凝胶剂对溃疡有明显的治疗作用。     

重组人粒细胞—巨噬细胞集落刺激因子的临床研究进展

本文着重从骨髓发育异常综合症，骨髓移植，外周血造血干细胞移植，恶性肿瘤放疗和化疗引起的白细胞减少症和白血病等方面介绍了重组人粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）近些年的临床研究进展情况。     

重组人粒细胞集落刺激因子的临床应用

本文回顾近年来相关文献,概述rhG-CSF的临床应用状况.该药可刺激骨髓细胞增加ANC、单核细胞、T淋巴细胞数量,增强ANC的吞噬作用,用于改善肿瘤、白血病患者放化疗后的ANC减少以及因粒细胞缺乏所合并的感染、骨髓移植后的造血恢复、外周血干细胞动员疗效确切.但有些肿瘤细胞能合成G-CSF,G-CSF也能促进某些肿瘤细胞的生长和转移.临床可根据个体情况选择恰当的用药时机、疗程、剂量,并进行药效监控,以达到合理用药.     

重组人粒细胞集落刺激因子致急性肾衰竭

1名54岁男性失代偿期肝硬化患者,皮下注射重组人粒细胞集落刺激因子(rhG-CSF)200μg,1次/d。用药后第2天患者出现尿色变深,第4天出现眼睑水肿,肉眼血尿、少尿等症状。BUN由4.8mmol/L升至7.9mmol/L(最高13.9mmol/L),Cr由113μmol/L升至154μmol/L(最高308μmol/L)。停用rhG-CSF,给予还原型谷胱甘肽、硫普罗宁、呋塞米等对症支持治疗。2周后肾功能恢复正常。     

重组人粒细胞巨噬细胞集落刺激因子喷雾剂的制备及药效研究

目的研制重组人粒细胞巨噬细胞集落刺激因子喷雾剂,探索其治疗溃疡、烧伤和烫伤的有效性。方法使用合理比例的保护剂、透皮吸收促进剂、抑菌剂和缓冲体系等,将重组人粒细胞巨噬细胞集落刺激因子制备成喷雾剂,通过动物药效实验探索其治疗溃疡、烧伤和烫伤的效果。结果重组人粒细胞巨噬细胞集落刺激因子喷雾剂通过2～8℃条件24个月长期稳定性考察,理化性质和生物活性没有明显变化。通过豚鼠溃疡模型和家兔烧伤和烫伤模型显示,药效非常显著。结论重组人粒细胞巨噬细胞集落刺激因子可制备成喷雾剂,为临床治疗溃疡、烧伤和烫伤等创面疾病提供了依据。     

国产重组人粒细胞集落刺激因子对造血系统的刺激作用

采用整体和离体两种方法,研究了国产重组人粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）对血血系统的影响,结果表明,国产ｒｈＧ－ＣＳＦ能减轻γ射线对小鼠外周血白细胞数的抑制,也能升高静注环磷酰胺恒河猴的外周血白细胞数,亦能直接刺激离体小鼠和人的骨髓细胞集落形成,说明国产品的作用性质和强度与进口品相似。     

重组人红细胞生成素中N-连接糖的毛细管凝胶电泳-激光诱导荧光测定法

目的：表征重组人红细胞生成素中影响体内生物学活性的N-连接糖谱。方法：以毛细管凝胶电泳-激光诱导荧光检测法(CGE-LIF)测定了重组人红细胞生成素(rHuEPO)中N-连接的多个糖基化形式,并以此为基础研究结构与活性的关系。结果：同一生产线上的样品,其N-连接糖的CGE-LIF图谱基本一致;不同表达载体所表达的rHuEPO,其N-连接糖的形式不同;活性不同的样品,各N-连接糖组分的相对含量存在很大差异。结论：建立的方法可用于判断产品的来源及批间差异,与肽图相结合可测定rHuEPO的一级结构。     

重组L-天门冬酰胺酶II的液相色谱/电喷雾离子化质谱法分析

目的　对基因重组L-天门冬酰胺酶II产品进行一级结构分析。方法　采用流动注射方式,电喷雾离子化质谱法测定分子量;三氯乙酸变性,胰蛋白酶水解后,HPLC测定肽图谱;结合质谱源内碰撞诱导解离技术,解析酶解肽段的氨基酸顺序。结果　分子量测定值与理论值不符,质谱解析表明样品中存在3个氨基酸变异现象,由此计算的分子量与测定值吻合。结论　液相色谱/电喷雾质谱法为基因重组蛋白质药物的一级结构分析和质量控制提供了新途径。     

A peptide targeted against phosphoprotein and leader RNA interaction inhibits growth of Chandipura virus – An emerging rhabdovirus

The fatal illness caused by Chandipura virus (CHPV), an emerging pathogen, presently lacks any therapeutic option. Previous research suggested that interaction between the virally encoded phosphoprotein (P) and the positive sense leader RNA (le-RNA) may play an important role in the viral lifecycle. In this report, we have identified a β-sheet/loop motif in the C-terminal domain of the CHPV P protein as essential for this interaction. A synthetic peptide encompassing this motif and spanning a continuous stretch of 36 amino acids (Pep_208–243) was found to bind the le-RNA in vitro and inhibit CHPV growth in infected cells. Furthermore, a stretch of three amino acid residues at position 217–219 was identified as essential for this interaction, both in vitro and in infected cells. siRNA knockdown-rescue experiments demonstrated that these three amino acid residues are crucial for the leader RNA binding function of P protein in the CHPV life cycle. Mutations of these three amino acid residues render the peptide completely ineffective against CHPV. Effect of inhibition of phosphoprotein–leader RNA interaction on viral replication was assayed. Peptide Pep_208–243 tagged with a cell penetrating peptide was found to inhibit CHPV replication as ascertained by real time RT-PCR. The specific inhibition of viral growth observed using this peptide suggests a new possibility for designing of anti-viral agents against Mononegavirale group of human viruses.     

Decoy receptor 3 (DcR3) is proteolytically processed to a metabolic fragment having differential activities against Fas ligand and LIGHT

Fas ligand (FasL) and Fas receptor are members of the tumor necrosis factor (TNF) receptor and ligand family that play an important role in regulating apoptosis in normal physiology. Decoy receptor 3 (DcR3) is a novel member of the TNF receptor superfamily, which binds to and blocks the activities of the ligands FasL and LIGHT. We have demonstrated that DcR3 was degraded rapidly to a major circulating metabolic fragment after subcutaneous administration in primates and mice. This fragment was also generated in subcutaneous tissue homogenate in vitro. Mass spectrometry and N-terminal sequencing indicated that DcR3 was proteolytically cleaved between R218 and A219 in the primary sequence to yield the fragment DcR3(1-218). While retaining its ability to bind LIGHT and inhibit LIGHT-mediated activities, DcR3(1-218) no longer bound FasL and did not inhibit FasL-mediated apoptosis in vitro. The primary sequence of DcR3 was molecularly engineered, changing the arginine residue at position 218 to glutamine to generate an analog, DcR3(R218Q), which we termed FLINT (LY498919). We demonstrated that FLINT was more stable to proteolytic degradation in vitro and in vivo and maintained its activity against both soluble FasL and soluble LIGHT in vitro. As a result, the modification in the sequence of DcR3 to produce FLINT (LY498919) should result in a pharmacologically superior molecule in the therapeutic intervention of diseases in which the pathogenesis is linked to FasL-mediated apoptotic or inflammatory events.     

On the biomarkers and mechanisms of konzo, a distinct upper motor neuron disease associated with food (cassava) cyanogenic exposure

Konzo is a self-limiting central motor-system disease associated with food dependency on cassava and low dietary intake of sulfur amino acids (SAA). Under conditions of SAA-deficiency, ingested cassava cyanogens yield metabolites that include thiocyanate and cyanate, a protein-carbamoylating agent. We studied the physical and biochemical modifications of rat serum and spinal cord proteins arising from intoxication of young adult rats with 50-200 mg/kg linamarin, or 200 mg/kg sodium cyanate (NaOCN), or vehicle (saline) and fed either a normal amino acid- or SAA-deficient diet for up to 2 weeks. Animals under SAA-deficient diet and treatment with linamarin or NaOCN developed hind limb tremors or motor weakness, respectively. LC/MS-MS analysis revealed differential albumin carbamoylation in animals treated with NaOCN, vs. linamarin/SAA-deficient diet, or vehicle. 2D-DIGE and MALDI-TOF/MS-MS analysis of the spinal cord proteome showed differential expression of proteins involved in oxidative mechanisms (e.g. peroxiredoxin 6), endocytic vesicular trafficking (e.g. dynamin 1), protein folding (e.g. protein disulfide isomerase), and maintenance of the cytoskeleton integrity (e.g. α-spectrin). Studies are needed to elucidate the role of the aformentioned modifications in the pathogenesis of cassava-associated motor-system disease.     

Identification of superoxide dismutase as a potential urinary marker of carbon tetrachloride-induced hepatic toxicity.

The aim of this study was the identification of a novel protein marker of hepatotoxicity in rat urine. Rats were dosed by gavage with carbon tetrachloride (CCl(4)) to induce acute liver injury. Surface enhanced laser desorption/ionisation (SELDI) ProteinChip technology revealed the appearance of a 15.7kDa protein in the CCl(4)-treated rat urine. One-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) identified an 18.4kDa protein in the CCl(4)-treated rat urine. The appearance of either protein was coincident over a time course during which they first appeared at 12h post-dosing, peaked at 36h and had disappeared again within 3 days post-dosing. The protein was identified by in-gel digestion and nano-electrospray (nano-ES)-tandem mass spectrometry as Cu/Zn superoxide dismutase (SOD-1). SOD activity was found to be increased by 61.4-fold in CCl(4)-treated rat urine. Western blots of tissue homogenates from the rats revealed a time-dependent loss of SOD-1 from the livers of CCl(4)-treated rats matching the time course of SOD-1 appearance in urine. SOD-1 is not specifically located in liver; however, its appearance in urine in response to acute CCl(4)-induced hepatotoxicity is a novel finding; this coupled with loss from the liver following injury suggests urinary SOD-1 may be a potential marker of hepatotoxicity.     

Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.     

Pharmaceutical applications of vibrational chemical imaging and chemometrics: a review

The emergence of chemical imaging (CI) has gifted spectroscopy an additional dimension. Chemical imaging systems complement chemical identification by acquiring spatially located spectra that enable visualization of chemical compound distributions. Such techniques are highly relevant to pharmaceutics in that the distribution of excipients and active pharmaceutical ingredient informs not only a product's behavior during manufacture but also its physical attributes (dissolution properties, stability, etc.). The rapid image acquisition made possible by the emergence of focal plane array detectors, combined with publication of the Food and Drug Administration guidelines for process analytical technology in 2001, has heightened interest in the pharmaceutical applications of CI, notably as a tool for enhancing drug quality and understanding process. Papers on the pharmaceutical applications of CI have been appearing in steadily increasing numbers since 2000. The aim of the present paper is to give an overview of infrared, near-infrared and Raman imaging in pharmaceutics. Sections 2 and 3 deal with the theory, device set-ups, mode of acquisition and processing techniques used to extract information of interest. Section 4 addresses the pharmaceutical applications.