Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

超短脉冲技术测量癌细胞荧光光谱与荧光寿命

研究用于癌症诊断与治疗的光敏剂血卟啉（hematoporphyrin derivative,HPD）的超快光动力学过程,采用超短脉冲激光光谱技术和皮秒时间相关单光子计数系统,测量经血卟啉培养的活体癌细胞与正常细胞的荧光光谱、荧光寿命特性及荧光峰值强度随时间变化曲线,并测量单一细胞内部不同位置的荧光寿命特性,观测到：癌细胞样品在645 nm处具有特有的光谱谱峰;癌细胞样品荧光寿命的快成分约150 ps慢成分约1200 ps,而正常细胞样品快成分约300 ps慢成分约2500 ps;癌细胞样品的荧光峰值强度经12小时衰减约10%,而正常细胞样品衰减约55%;在细胞内部荧光寿命300 ps的快成分十分显著,且中心部位血卟啉浓度最高.癌细胞与正常细胞的荧光光谱、荧光寿命特性及荧光峰值强度随时间变化曲线相差十分明显,反映了癌细胞与正常细胞对血卟啉亲和特性有显著的差异,测量结果确认了荧光光谱技术诊断与治疗癌症的可行性,并对发展超短脉冲激光光谱技术早期诊断与治疗癌症具有重要的指导意义和临床应用价值.     

Alanine scanning mutational analysis of the ligand binding pocket of the human Vitamin D receptor

We achieved exhaustive alanine scanning mutational analysis of the amino acid residues lining the ligand binding pocket of the Vitamin D receptor to investigate the mechanism of the ligand recognition by the receptor. This is the first exhaustive analysis in the nuclear receptor superfamily. Our results demonstrated the role and importance of all the residues lining the ligand binding pocket. In addition, this analysis was found to indicate ligand-specific ligand-protein interactions, which have key importance in determining the transactivation potency of the individual ligands. Thus, the analysis using 1beta-methyl-1alpha,25-dihydroxyvitamin D(3) revealed the specific van der Waals interactions of 1beta-methyl group with the receptor.     

Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability

Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.     

Identification of a novel, highly variable amino-terminal amino acid sequence element in the nuclear intermediate filament protein lamin B(2) from higher vertebrates

By comparing newly available cDNA sequences of the human intermediate filament protein lamin B(2) with published sequences, we have identified an additional translation initiation codon 60 nucleotides upstream of the previously assumed translation start. In addition, corresponding sequences were identified in the chimpanzee, mouse, rat and bovine genes and cDNAs, respectively. Therefore, we generated antibodies against these potential 20 new amino acids of the human sequence. By immunoblot analysis and immunofluorescence microscopy we show that human lamin B(2) is indeed synthesized as a longer version than previously reported, because it contains these additional 20 amino acids. Notably, the sequence homology to mouse, rat and bovine lamin B(2) is significantly lower in this segment than in that between the second methionine codon and the start of the alpha-helical rod indicating that the tip of the "head" is engaged in more species-specific functions. Forced expression of the GFP-tagged authentic "long" and the 20 amino acid shorter version of lamin B(2) in human cultured SW-13 cells demonstrated that both the longer and the shorter version are properly integrated into the nuclear lamina, although the shorter version exhibited a tendency to disturb envelope architecture at higher expression levels.     

Q开关激光爆破技术对豚鼠皮肤色素的影响

目的研究Q开关激光爆破术对豚鼠黑素细胞的影响及照射周边组织的变化,为临床治疗皮肤色素病变提供实验依据。方法用Q开关-YAG激光分别照射豚鼠黑色毛区及棕色毛区(波长分别用1064nm和530nm,光斑直径2mm),实验动物20只,随机分四组,分别于照射后间隔7d、10d、14d取材,照射前取材作对照,10%甲醛固定,冰冻切片,分别用HE和DOPA反应显示黑素细胞。结果照射后皮肤黑色素颗粒逐渐减少至消失,照射后30d黑毛区与棕毛区肉眼见照射区皮肤变白,毛也变白,HE染色皮肤、毛囊及毛未见黑色素颗粒,DOPA反应表皮黑色素细胞、毛囊和毛均呈阴性反应,部分豚鼠棕毛区毛及毛囊见黄色色素颗粒。结论波长1064nm和532nm Q-YAG激光对豚鼠皮肤黑色素细胞和黑色素颗粒的破坏效果显著;但对棕色色素清除效果较差。波长1064nm Q-YAG激光对豚鼠皮肤黑色素消减与照射次数有关,与照射间隔时间长短无关。     

翠绿宝石激光脱毛350例疗效分析

目的:评价翠绿宝石激光脱毛治疗的疗效。方法:使用波长为755 nm的GentleLASE Plus激光对350例门诊患者的不同部位的体毛进行多次治疗,并按不同部位和治疗次数进行分组,分析其疗效。结果:经过多次治疗后,总体治愈率为83.71%,疗效与不同的部位有关,且与治疗次数呈正相关。其中5例出现暂时性色素沉着。结论:使用GentleLASE Plus激光进行脱毛治疗效果良好,并发症少,是目前较为理想的脱毛治疗方法。     

Amphipathic Helical Cationic Antimicrobial Peptides Promote Rapid Formation of Crystalline States in the Presence of Phosphatidylglycerol: Lipid Clustering in Anionic Membranes

Five AHCAPs exhibiting a broad-spectrum of antimicrobial activity, were examined with regard to their action in lipid mixtures with two anionic lipids, PG and CL. We find that all of the peptides studied were capable of promoting the formation of crystalline phases of DMPG in mixtures of DMPG and CL, without prior incubation at low temperatures. This property is indicative of the ability of these peptides to cluster CL away from DMPG. In contrast, the well studied antimicrobial cationic peptide magainin 2 does not cluster anionic lipids. We ascribe the lower anionic lipid clustering ability of magainin to its low density of positive charges compared with the five other AHCAPs used in this work. The peptide MSI-1254 was particularly potent in segregating these two anionic lipids. Consequently, clusters enriched in DMPG appear in a lipid mixture with CL. These can rapidly form higher temperature crystalline phases because of the increased permeability of the bilayer caused by the AHCAPs. The polyaminoacids, poly-L-Lysine and poly-l-arginine are also very effective in causing this segregation. Thus, the clustering of anionic lipids by AHCAPs is not confined only to mixtures of anionic with zwitterionic lipids, but it extends to mixtures containing different anionic headgroups. The resulting effects, however, have different consequences to the biological activity. This finding broadens the scope for which an AHCAP agent will cluster lipids in a membrane.     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.