Rapid Fluorescein and Protein Assay Method for Fluorescent-Antibody Conjugates

A method is presented for the rapid determination of the fluorescein content, protein content, and fluorescein-to-protein ratio for immune globulin conjugates with fluorescein isothiocyanate as the fluor. This method is based on the absorbance of the fluorescent antibody at those wavelengths primarily associated with the fluorescein and gamma-globulin fractions, and permits these materials to be determined by a single nondestructive analytical procedure. A small sample of the fluorescent antibody, in many cases 0.1 ml or less, is adequate for the above determinations. A nomograph is presented which allows simultaneous determination of the materials from the observed absorbance at the two wavelengths. The method is sufficiently accurate for most applications of the fluorescent-antibody techniques. Although this procedure has been developed primarily for fluorescent-antibody conjugates prepared from rabbit gamma-globulin, it can be used directly for antibodies prepared from other animals provided the gamma-globulin is relatively free from albumin.     

Conjugates of Chitinase with Fluorescein Isothiocyanate or Lissamine Rhodamine as Specific Stains for Chitin In Sztu

The fluorescent chitinase technique is based on the specific affinity of the enzyme for its substrate and applicable when an enzyme can be coupled with a fluorescent dye. Fluorescent chitinase specifically stained chitinous structures in several fungi and an insect, but failed to stain other polysaccharides in bacterial and algal cell walls. Freezing-microtome sections of Drosophila and fungal mycelia 6 μ thick were fixed in acetone for 5 min, then stained and mounted in fluorescent chitinase. Staining of smears of unsectioned fungal material required 5 min in absolute acetone, 5 min in 95% ethanol-1 N aqueous acetic acid (1:1), 10 min in 0.2 M phosphate buffer, PH 5.7, 1 sec in enzyme-dye conjugate, and 10 min in carbonate-bicarbonate buffer (0.2 M, pH 10.7, for chitinase-FITC; pH 7.6, for chitinase-LRBC). Preparations are viewed microscopically with ultraviolet light.     

Controlled Fabrication of Gold Nanoparticle and Fluorescent Protein Conjugates

The unique optical properties of gold nanoparticles make them attractive for a wide range of applications which require optical detection and manipulation techniques. Here, we experimentally demonstrate the use of single femtosecond pulses at resonance wavelength for a controlled conjugation of gold nanoparticles and fluorescent proteins. This optically driven reaction is rigorously studied and analyzed using a variety of experimental techniques, and a detailed model is proposed which describes the adsorption of the proteins onto the nanoparticles' surface, as well as their subsequent desorption by a reducing agent. Potential applications of the resulting nanoparticle?Cprotein conjugates include controlled delivery of fluorescent markers and local sensing of biochemical processes.     

Evidence that TP_0144 of Treponema pallidum Is a Thiamine-Binding Protein

Thiamine pyrophosphate (TPP), the biologically active form of thiamine (also known as vitamin B₁), is an essential cofactor for several important enzymes involved in carbohydrate metabolism, and therefore, it is required for all living organisms. We recently found that a thiamine-binding protein (TDE_0143) is essential for the survival of Treponema denticola, an important bacterial pathogen that is associated with human periodontitis. In this report, we provide experimental evidence showing that TP_0144, a homolog of TDE_0143 from the syphilis spirochete Treponema pallidum, is a thiamine-binding protein that has biochemical features and functions that are similar to those of TDE_0143. First, structural modeling analysis reveal that both TDE_0143 and TP_0144 contain a conserved TPP-binding site and share similar structures to the thiamine-binding protein of Escherichia coli. Second, biochemical analysis shows that these two proteins bind to TPP with similar dissociation constant (K_d) values (TDE_0143, K_d of 36.50 nM; TP_0144, K_d of 32.62 nM). Finally, heterologous expression of TP_0144 in a ΔTDE_0143 strain, a previously constructed TDE_0143 mutant of T. denticola, fully restores its growth and TPP uptake when exogenous thiamine is limited. Collectively, these results indicate that TP_0144 is a thiamine-binding protein that is indispensable for T. pallidum to acquire exogenous thiamine, a key nutrient for bacterial survival. In addition, the studies shown in this report further underscore the feasibility of using T. denticola as a platform to study the biology and pathogenicity of T. pallidum and probably other uncultivable treponemal species as well.     

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.     

Synthesis of a New Vitamin D3 Hapten and Its Protein Conjugates

Russian Journal of Bioorganic Chemistry - The synthesis of 25-(1-carboxymethoxy)-imino-3(S)-hydroxy-9,10-seco-27-norcholesta-5(Z),7(E),10(19)-triene, a new hapten of vitamin D3, has been carried...     

Effect of Low Charge and High Hydrophobicity on Antimicrobial Activity of the Quinazolinone-Peptide Conjugates

Peptides of relatively low charge and a high number of hydrophobic amino acids were designed. The amino acid sequence of designed peptides was GXGVP, where X equaled to W, Y, F, D, and T with a combination of hydrophobic, charged and polar units. These peptides were linked to quinazolinones to obtain a new class of compounds with synergistic features. The hybrids displayed antimicrobial activity against Gram-positive and Gram-negative bacteria. In particular, Trp, Tyr, and Phe-containing peptides showed greater antimicrobial potency than the reference standards. Alkyl chain length variations in heterocyclic moiety indicated that hybrids with propyl group were more active than butyl derivatives. Improved results were observed for debenzylated versions of the conjugates compared to their benzylated counterparts. Implementation of the hybrid structures of varying charge, hydrophobicity, and alkyl chain length would be a promising approach to obtaining effective antimicrobial agents.     

Intracellular turnover of Fluorescein diacetate. Influence of membrane ionic gradients on Fluorescein efflux

Summary The influence of the membrane ionic gradient on the efflux of Fluorescein after intracellular turnover of Fluorescein diacetate was studied in HeLa cells. The kinetics of Fluorescein efflux was monitored by determining with flow cytometry the decrease in fluorescence intensity of single cells. Alterations of the Na⁺ and K⁺ gradients were induced experimentally by using otiabain, ionophores or buffers in which the ion concentration was modified. The sodium gradient was also altered by using Na⁺-co-transported amino acids. Independent evidence of these changes was obtained with a potential-sensitive indicator, 3,3′-dihexyloxacarbocyanine iodide. Conditions inducing a reduction or dissipation of the ionic gradients caused a decrease in the rate constant of Fluorescein release. In contrast, enhancement of the gradients increased the efflux rate. These results indicate that the release of Fluorescein from living cells is influenced by the membrane potential. Thus, the turnover of Fluorescein diacetate may provide a useful technique for assessing changes in membrane permeability properties related to tomic gradients.     

Diversity of Bisubstrate Binding Modes of Adenosine Analogue-Oligoarginine Conjugates in Protein Kinase A and Implications for Protein Substrate Interactions

Crystal structures of the catalytic subunit α of cAMP-dependent protein kinase (PKAc) with three adenosine analogue-oligoarginine conjugates (ARCs) are presented. The rationally designed ARCs include moieties that, in combination, target both the ATP- and the peptide-substrate-binding sites of PKAc, thereby taking advantage of high-affinity binding interactions offered by the ATP site while utilizing an additional mechanism for target specificity via binding to the peptide substrate site. The crystal structuresdemonstrate that, in accord with the previously reported bisubstrate character of ARCs, the inhibitors occupy both binding sites of PKAc. Further, they show new binding modes that may also apply to natural protein substrates of PKAc, which have not been revealed by previous crystallographic studies. The crystal structures described here contribute to the understanding of the substrate-binding patterns of PKAc and should also facilitate the design of inhibitors targeting PKAc and related protein kinases.     

SARS冠状病毒核壳蛋白对蛋白翻译的影响

目的:研究SARS冠状病毒核壳蛋白(N蛋白)对蛋白翻译的影响。方法:构建N蛋白表达载体FLAG-pcDNA3-N,分别与FLAG-pcDNA3和表达荧光素酶的质粒共转染293T人胚肾细胞,通过检测荧光素酶的活性来判断N蛋白对细胞内蛋白翻译的影响;在体外翻译体系中检测N蛋白对体外翻译的影响。结果:构建了载体FLAG-pcDNA3-N,在293T人胚肾细胞内表达后,荧光素酶活性被抑制;在体外翻译体系中加入N蛋白,体外翻译被抑制。结论:SARS冠状病毒N蛋白抑制蛋白翻译。     

梅毒螺旋体重组抗原的表达及在梅毒血清学诊断中的应用

目的：克隆、表达梅毒螺旋体（Treponema pallidum, Tp）膜蛋白TpN47、TpN17、TpN44.5和TpN15,建立双抗原夹心ELISA法,探讨其在梅毒血清学诊断中的应用。方法：应用聚合酶链反应法（PCR）从Tp全基因组中扩增4个目的片段,克隆到载体pET-HT-JKM中,在BL21(DE3)plysS菌中诱导表达,Ni-NTA亲和层析法纯化重组蛋白,辣根过氧化物酶标记,双抗原夹心酶联免疫吸附法检测人血清中的特异性IgM和IgG抗体。结果：成功表达了4种重组蛋白用作ELISA包被和酶标抗原检测国家Tp参比血清,特异性和灵敏度均为100％;检测385份临床血清样本,结果与TPPA法相比符合率达到99％（P＜0.01）,灵敏度和特异性分别为98.2%（162/165）和99.5%（219/220）。结论：表达的4种重组抗原具有很好的生物活性,为理想的梅毒的血清学诊断用抗原。以这4种表达蛋白为基础建立的双抗原夹心ELISA法灵敏度高,特异性好,而且方法简单,结果客观,易于自动化操作,值得临床大力推广。     

Identification of the Treponema pallidum subsp. pallidum glycerophosphodiester phosphodiesterase homologue

To identify potential opsonic targets of Treponema pallidum subsp. pallidum, a treponemal genomic expression library was constructed and differentially screened with opsonic and non-opsonic T. pallidum antisera. This method identified an immunoreactive clone containing an open reading frame encoding a 356 residue protein. Nucleotide sequence analysis demonstrated the translated protein to be a homologue of glycerophosphodiester phosphodiesterase, a glycerol metabolizing enzyme previously identified in Haemophilus influenzae, Escherichia coli, Bacillus subtilis and Borrelia hermsii. Sequence alignment analyses revealed the T. pallidum and H. influenzae enzymes share a high degree of amino acid sequence similarity (72%), suggesting that in T. pallidum this molecule may be surface exposed and involved in IgD binding as is the case with its counterpart in H. influenzae.