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1.
The problem of the simultaneous use in flow cytometry of N greater than 2 antibodies in conjunction with two fluorochromes was investigated. Theoretical analysis led to a labeling procedure and reconstruction formula that allow N-dimensional labeling distributions to be obtained from two-dimensional fluorescence distributions. The general problem of M greater than or equal to 2 fluorochromes and N greater than M antibodies was shown to be reducible to the case of two fluorochromes. The method was tested by a triple labeling analysis of murine thymocytes.  相似文献   

2.

Background

The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development.

Methods

A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy

Results

Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1.

Conclusion

A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.  相似文献   

3.
4.
Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1) selection of fluorescent dyes having minimal spectral overlap is complicated, and 2) quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biotène PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; α=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p<0.05) on both resin composites, whereas LTO did not produce differences (p>0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.  相似文献   

5.
Well-known dynamic programming algorithms exist for comparing two finite sequences inO(N 2) time and storage, whereN is the common sequence length. Extensions to the comparison ofM finite sequences requireO((2N) M) time and storage, making such algorithms difficult even forM=3. A simple generalization of the sequences makes it possible to obtain some results about the geometry of sequence alignments. These ideas suggest heuristic approaches to problems of comparing several sequences. IfM sequences are known to be related by a binary tree, they can be aligned inO(MN 2) time andO(N 2+NM) storage. This work was supported by a grant from the System Development Foundation.  相似文献   

6.
Lung N-oxidase enzyme activity was about three times higher than liver N-oxidase at the pH optimum, about pH 8.9, whereas the activities were nearly the same at more physiological ranges of pH. The lung N-oxidase was also stimulated about 2-fold by 100 mM Mg2+ and by 0.1 mM Hg2+, whereas liver N-oxidase activity was inhibited by these concentrations of ions. The difference in response of liver and lung enzymes to Mg2+ and Hg2+ was not altered by preparing the microsomes in the presence of 50 mM ethylenediamine tetraacetic acid (EDTA) in 0.1 M Tris (hydroxymethyl) amino methane (Tris) buffer or 50 mM EDTA in 0.1 M KPO4 buffer, both at pH 7.6, indicating that the differences are probably not due to the presence of endogenous metals. The difference between the liver and lung N-oxidase systems may be due to the tissue environment rather than to the enzyme itself since mercury stimulation of lung N-oxidation began to disappear upon partial purification of the N-oxidase enzymes. In contrast to the effects of Hg2+ and Mg2+, 1 mM Ni2+ enhanced liver N-oxidase activity about 30% and 5 mM Ni2+ stimulated lung enzyme activity about 30% whereas concentrations above 10 mM were inhibitory to both N-oxidases. Both liver and lung demethylase activities were inhibited by these concentrations of Mg2+, Hg2+ and Ni2+.Various suifhydryl reagents were also tested for their effects on these enzymes. The mercurials, para-chloromercurybenzoate (pCMB) and phenylmercuryacetate (PMA) at concentrations of 0.1 mM had the same effect as HgCl2 inhibiting both demethylases and liver N-oxidase, but stimulating lung N-oxidase activity. However, 0.1 mM to 1 mMN-ethylmaleimide (NEM) and iodoacetamide had little if any effect on either liver or lung N-oxidase. It was also shown that Hg2+ effects on N-oxidase activity could be overcome by dilution.Changes in N,N-dimethyl aniline (DMA) metabolism with age were followed in rabbits from 4 days old to adult. There was a steady increase in lung demethylase activity and N-oxidase activity in the liver and lung to adult levels. However, the liver demethylase had a sharp increase in activity between 2 weeks and 1 month much like that seen with benzphetamine demethylase in rabbit liver.Activities of N-demethylase in liver and lung, and N-oxidr.se in liver from new-born rabbits were from 10 to 20 % of adult levels. However, in lung, N-oxidase activities in the newborn were about 50 % of adult levels. Microsomal N-oxidation in lungs from 2-day-old rabbits was stimulated by 0.1 mM mercury just as in the adult.  相似文献   

7.

Background

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters.

Methodology/Principal Findings

To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a TH1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model.

Conclusions

Even though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci.  相似文献   

8.
SCP2 and SCP3 are major protein components of the lateral elements (LEs) of synaptonemal complexes (SCs) of the rat, with M rs of 173,000 and 30,000. We performed a detailed immunocytochemical comparison of the localization of SCP2 and SCP3 within SCs at the electron microscopic level. The ultrastructural localization of SCP2 and SCP3 was analyzed by immunogold labeling of two types of preparations, namely surface-spread spermatocytes and ultrathin sections of Lowicryl-embedded testicular tissue of the rat. For each of the antisera used, the distribution of immunogold label over SCs in surface-spread spermatocytes differed significantly from the distribution of label on sections. We attributed this difference to artifacts caused by the surface-spreading technique, and therefore we relied on sections for the precise localization of epitopes. On sections, the distribution of label obtained with two antisera against nonoverlapping, widely separated fragments of SCP2 did not differ significantly. There was a small but significant difference between the labeling pattern obtained with an anti-SCP3 serum and the pattern obtained with either of the two antisera against fragments of SCP2; although for all three antisera the peak of the immunogold label coincided with the center of the LE, the distributions of label obtained with the antisera against fragments of SCP2 were asymmetrical, with a shoulder at the inner side of the LE, whereas the distribution of label obtained with anti-SCP3 serum was symmetrical. Furthermore, we observed fuzzy connections between the LEs that were labeled by anti-SCP2 but not anti-SCP3 antibodies. It is possible that labeling of these fuzzy bridges caused the shoulder in the gold label distributions obtained with anti-SCP2 antibodies. Received: 25 June 1998; in revised form: 4 August 1998 / Accepted: 7 August 1998  相似文献   

9.
Which bacterial taxonomic groups can be used in monitoring saline water methanol-utilizing denitrification and whether nitrate is transformed into N2 in the process are unclear. Therefore, methylotrophic bacterial communities of two efficiently functioning (nitrate/nitrite reduction was 63–96 %) tropical and cool seawater reactors at a public aquarium were investigated with clone library analysis and 454 pyrosequencing of the 16S rRNA genes. Transformation of nitrate into N2 was confirmed using 15N labeling in incubation of carrier material from the tropical reactor. Combining the data with previous study results, Methylophaga and Hyphomicrobium were determined to be suitable target genera for monitoring the function of saline water methanol-fed denitrification systems. However, monitoring was not possible at the single species level. Interestingly, potential nitrate-reducing methylotrophs within Filomicrobium and closely related Fil I and Fil II clusters were detected in the reactors suggesting that they also contributed to methylotrophic denitrification in the saline environment.  相似文献   

10.
TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds ofTorresea acreana, one of the two known species ofTorresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears asM r 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2,Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.  相似文献   

11.
Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.  相似文献   

12.
The presence of chitin in hyphal cell walls and regenerating protoplast walls ofSaprolegnia monoi¨ca was demonstrated by biochemical and biophysical analyses. α-Chitin was characterized by X-ray diffraction, electron diffraction, and infrared spectroscopy. In hyphal cell walls, chitin appeared as small globular particles while cellulose, the other crystalline cell wall component, had a microfibrillar structure. Chitin synthesis was demonstrated in regenerating protoplasts by the incorporation of radioactiveN-acetylglucosamine into a KOH-insoluble product. Chitin synthase activity of cell-free extracts was particulate. This activity was stimulated by trypsin and inhibited by the competitive inhibitor polyoxin D (Ki 20 μM). The reaction product was insoluble in 1M KOH or 1M acetic acid and was hydrolyzed by chitinase into diacetylchitobiose. Fungal growth and cell wall chitin content were reduced when mycelia were grown in the presence of polyoxin D. However, hyphal morphology was not altered by the presence of the antibiotic indicating that chitin does not seem to play an important role in the morphogenesis ofSaprolegnia.  相似文献   

13.
Treponema denticola ATCC 35405, a human oral spirochete associated with periodontal disease, was shown to contain three enzymes (I, II, and III) with proline iminopeptidase activity. II and III were considered to be true iminopeptidases, whereas enzyme I was found to be a benzoylarginine peptidase with iminopeptidase activity. Enzyme III, the dominant proline iminopeptidase ofT. denticola in terms of its activity towardN-l-prolyl-2-naphthylamine, was considered to be a sulfhydryl peptidase: 0.167 M p-chloromercuribenzoic acid totally inactivated the enzyme, and 1.0 mM dithiothreitol restored 92% of activity. The activity of this enzyme was not affected by metal chelators. Chemical modification of enzyme III suggests that tyrosyl (or histidyl) and carboxyl groups may be necessary for its activity. The hydrolysis ofN-l-prolyl-2-naphthylamine was found to be very characteristic ofT. denticola ATCC 35405; out of 24 differentN-l-aminoacyl-2-naphthylamines tested, only the proline derivative was hydrolyzed at a high rate. The substrate specificity of the enzymes discovered indicates that they may be important for the nutrition ofT. denticola. The iminopeptidase activity may be related to the pathogenicity of this organism in periodontal disease.  相似文献   

14.
We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose or [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-β-N-acetylglucosaminidase H, which specifically cleaves the ‘high-mannose’ class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing.This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a ‘high-mannose’ oligosaccharide precursor and a ‘complex’ carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-β-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistent to endo-β-N-acetylglucosaminadase H cleavage. The final ‘complex’ or processed oligosaccharide structure contained approximately two-thirds less associated with the mature glycoprotein. They also indicate that the ‘complex’ structure is synthesized as a ‘high-mannose’ intermediate which is processed by the removal of mannose.  相似文献   

15.
Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.  相似文献   

16.
Cytokinins, which have some structural similarities to ancymidol, a plant growth retardant, were tested for their effects on the cell-free oxidation ofent-kaurene. Results indicate that several cytokinins inhibit this reaction in microsomal extracts of liquid endosperm from immature wild cucumber seeds. N6-cyclohexanemethyladenine was the most active (inhibiting 50% of the controlent-kaurene oxidation at 2×10?6 M). N6-isoamyladenine, N6-benzyladenine, N6-(Δ2-isopentenyl)adenine and dihydrozeatin were active at successively higher concentrations. Zeatin, kinetin, adenine, N6-benzyladenosine, and N6-(isopentenyl)adenosine were inactive in this system. The basis for the inhibition ofent-kaurene oxidation by cytokinins may be similar to that of ancymidol: interaction with cytochrome P-450. A binding spectrum similar to that of ancymidol with cytochrome P-450 from wild cucumber endosperm microsomes was obtained with four active cytokinins. The cytokinin binding properties of this protein are currently under investigation. No metabolism of N6-benzyladenine could be detected under conditions in which the cytokinin inhibited the oxidation ofent-kaurene toent-kaurenol.  相似文献   

17.

Background

The interaction of environmental chemicals and their metabolites with biological macromolecules can result in cytotoxic and genotoxic effects. 4-Aminobiphenyl (4-ABP) and several other related arylamines have been shown to be causally involved in the induction of human urinary bladder cancers. The genotoxic and the carcinogenic effects of 4-ABP are exhibited only when it is metabolically converted to a reactive electrophile, the aryl nitrenium ions, which subsequently binds to DNA and induce lesions. Although several studies have reported the formation of 4-ABP-DNA adducts, no extensive work has been done to investigate the immunogenicity of 4-ABP-modified DNA and its possible involvement in the generation of antibodies in bladder cancer patients.

Methodology/Principal Findings

Human DNA was modified by N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), a reactive metabolite of 4-ABP. Structural perturbations in the N-OH-AABP modified DNA were assessed by ultraviolet, fluorescence, and circular dichroic spectroscopy as well as by agarose gel electrophoresis. Genotoxicity of N-OH-AABP modified DNA was ascertained by comet assay. High performance liquid chromatography (HPLC) analysis of native and modified DNA samples confirmed the formation of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4ABP) in the N-OH-AABP damaged DNA. The experimentally induced antibodies against N-OH-AABP-modified DNA exhibited much better recognition of the DNA isolated from bladder cancer patients as compared to the DNA obtained from healthy individuals in competitive binding ELISA.

Conclusions/Significance

This work shows epitope sharing between the DNA isolated from bladder cancer patients and the N-OH-AABP-modified DNA implicating the role of 4-ABP metabolites in the DNA damage and neo-antigenic epitope generation that could lead to the induction of antibodies in bladder cancer patients.  相似文献   

18.
The in vitro labeling and stability of 99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the 99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The 99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the 99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between 99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N2S2) compound. The Fab′ with IgG2a isotype displayed tighter binding to 99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N2S2 challenge and incubation with human plasma. The in vivo biodistribution of five 99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable 99mTc labeling of antibody fragments from three of the major murine isotypes.  相似文献   

19.
We report here the isolation and molecular characterization of single-chain Fv (scFv) antibodies raised against two major allergens, Cryj1 and Cryj2, in the pollen of Cryptomeria japonica by the phage display method. Recombinant phages that produced scFv antibodies that bound to Cryj1 or Cryj2 were isolated by selection with immobilized antigens in microtiter plates. After selection of six Cryj1- and four Cryj2-specific scFv antibodies with strong binding activity, we performed pairwise interaction analysis of them by surface plasmon resonance. The analysis revealed that the scFv antibodies against Cryj1 bound to only four non-overlapping epitopes, with dissociation constants that ranged from 4.84×10?9 M to 1.62×10?7 M. By contrast, four Cryj2-specific scFv antibodies inhibited each other’s binding to Cryj2, with dissociation constants from 1.11×10?7 M to 4.21×10?7 M. Our results indicate that recombinant technology provides a time-saving method for the production of antibodies against pollen allergens.  相似文献   

20.
In short, the model consists of a two-dimensional set of edge detecting units, modelled according to the zero-crossing detectors introduced first by Marr and Ullman (1981). These detectors are located peripherally in our synthetic vision system and are the input elements for an intelligent recurrent network. The purpose of that network is to recognize and categorize the previously detected contrast changes in a multi-resolution representation of the original image in such a manner that the original information will be decomposed into a relatively small numberN of well-defined edge primitives. The advantage of such a construction is that time-consuming pattern recognition has no longer to be done on the originally complex motion-blurred images of moving objects, but on a limited number of categorized forms. Based on a numberM of elementary feature attributes for each individual edge primitive, the model is then able to decompose each edge pattern into certain features. In this way anM-dimensional vector can be constructed for each edge. For each sequence of two successive frames a tensor can be calculated containing the distances (measured inM-dimensional feature space) between all features in both images. This procedure yields a set ofK—1 tensors for a sequence ofK images. After cross-correlation of allN ×M feature attributes from image (i) with those from image (i+1), wherei = 1, ...,K - 1, probability distributions can be computed. The final step is to search for maxima in these probability functions and then to construct from these extremes an optimal motion field. A number of simulation examples will be presented.  相似文献   

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