Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. An example is natalizumab, a recombinant IgG4 antibody directed against α4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus, IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions.     

A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway

Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H(2)O/25% NH(3). Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.     

The activity of Triton X-100 soluble chlorophyllase in liposomes

Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) was isolated and purified from Phaseolus vulgaris L. chloroplasts and etioplasts dissolved in 1% Triton X-100 and 10% glycerol. A 100 and 40-fold purification, respectively, was achieved. Enzyme preparations from both sources had similar affinities for chlorophyll a when assayed in a Triton X-100 medium. When electrophoresed in sodium dodecyl sulphate polyacrylamide gels the major band in both preparations migrated as a peptide of 30,000 daltons. Chlorophyll containing liposomes were also used as a substrate for chlorophyllase. The rate of hydrolysis did not follow Michaelis-Menten kinetics. When chlorophyllide a or methyl chlorophyllide a was incorporated in the liposomes, then in the presence of phytol dissolved in methanol, methylchlorophyllide a and chlorophyll a were shown to be synthesized. Apparently the purified enzyme in the presence of lipids, is endowed with both synthetic and hydrolytic activity.Abbreviations DEAE diethylaminoethyl - MeOH methanol - SDS sodium dodecyl sulphate     

Heterologous expression,purification, and enzymatic characterization of the acyclic carotenoid 1,2-hydratase from Rubrivivax gelatinosus

The carotenoid 1,2-hydratase CrtC from Rubrivivax gelatinosus has been expressed in Escherichia coli in an active form and purified by affinity chromatography. The enzyme catalyzes the conversion of various acyclic carotenes including 1-hydroxy derivatives. This broad substrate specificity reflects the participation of CrtC in 1'-HO-spheroidene and in spirilloxanthin biosynthesis. Enzyme kinetic studies including the determination of substrate specificity constants indicate that among the alternative biosynthetic routes to 1'-HO-spheroidene the one via spheroidene is the dominating pathway. In contrast to CrtC from Rvi. gelatinosus, the equivalent enzyme from Rhodobacter capsulatus, a closely related bacterium which lacks the biosynthetic branch to spirilloxanthin and accumulates spheroidene instead of substantial amounts of 1'-HO-spheroidene, is extremely poor in converting 1-HO-carotenoids. The individual catalytic properties of both carotenoid 1,2-hydratases reflect the in situ carotenogenic pathways in both purple photosynthetic bacteria.     

Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts

Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs.     

Export of porin to the outer membrane of the phototrophic bacterium Rhodobacter capsulatus 37B4

Export of porin to the outer membrane of the phototrophic purple bacterium Rhodobacter capsulatus was studied with the use of the uncoupler of the electron transport chain, carbonylcyanide-m-chlorophenylhydrazone (CCCP). The agent reversibly blocked the transport of porin across the cytoplasmic membrane. By means of radioactive labeling and immunoprecipitation, porin was found to occur in two forms: (i) the exported form that was extractable from the outer membrane without disrupting the cells, and (ii) a pre-form with a slightly higher apparent molecular mass which accumulated in the cells during the block of the export process. Proteolysis studies revealed that the preform was highly sensitive to added proteases, whereas the exported form was resistant.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - OMP outer membrane porin; pre-OMP, form of outer membrane porin before export - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

Binding protein dependent transport of C4-dicarboxylates in Rhodobacter capsulatus

The characteristics of malate transport into aerobically grown cells of the purple photosynthetic bacterium Rhodobacter capsulatus were determined. A single transport system was distinguished kinetically which displayed a K_t value of 2.9 ± 1.2 μM and V_max of 43 ± 6 nmol · min^-1 · mg^-1 protein. Competition experiments indicated that the metabolically related C4-dicarboxylates succinate and fumarate are also transported by this system. Malate uptake was sensitive to osmotic shock and evidence from the binding of radiolabelled malate and succinate to periplasmic protein fractions indicated that transport is mediated by a dicarboxylate binding protein. The activity of the transport system was studied as a function of external and internal pH and it was found that a marked activation of uptake occurred at intracellular pH values greater than 7. The use of a high affinity binding protein dependent system to transport a major carbon and energy source suggests that Rhodobacter capsulatus would be capable of obtaining growth sustaining quantities of C4-dicarboxylates even if these were present at very low concentrations in the environment.     

Sequence analysis and interposon mutagenesis of a sensor-kinase (DctS) and response-regulator (DctR) controlling synthesis of the high-affinity C4-dicarboxylate transport system in Rhodobacter capsulatus

Summary A two-component sensor-regulator system has been identified in the purple photosynthetic bacterium Rhodobacter capsulatus, which controls the expression of high-affinity C4-dicarboxylate transport activity in these cells. Nucleotide sequencing has revealed the existence of two genes, dctS and dctR, which together form an operon linked to, but divergently transcribed from, the previously identified dctP gene, which encodes the periplasmic binding protein of the transport system. The DctS protein is predicted to be a membrane-bound sensor-kinase with two potential membrane-spanning sequences in the N-terminal region. DctR was found to have sequence similarity throughout its entire length with proteins in the FixJ subfamily of response-regulators, especially to FixJ itself (42% identical residues). Insertional inactivation of the dctS and dctR genes resulted in the inability of the resulting mutants to grow on or transport malate, succinate or fumarate under aerobic conditions in the dark, and such mutants did not express the DctP protein. The mutants were complemented in trans by plasmids containing intact copies of the dctS and dctR genes.     

Nitrogenase and photosynthetic activities of chemostat cultures of Rhodobacter capsulatus 37b4 grown under different illuminations

Nitrogen fixation as well as structural and functional properties of the photosynthetic apparatus were studied with phototrophically grown chemostat cultures of Rhodobacter capsulatus strain 37b4. Illumination was varied between 3,000 and 30,000 lx at a constant dilution rate of D=0.075 h^-1. Steady state parameters of growth revealed two forms of limitation, i.e. energy limitation in the range of 3,000 to about 10,000 lx and nitrogen limitation at higher illuminations. Over the entire range of illumination, the specific bacteriochlorophyll content and the amount of total bacteriochlorophyll per photochemical reaction center remained essentially constant. Photophosphorylation activity remained constant up to 20,000 lx but was slightly increased at 30,000 lx. Hydrogen evolution and acetylene reduction activities of cellular nitrogenase were assayed under saturating light conditions with samples taken from cultures growing under steady state conditions. In spite of the apparent constancy of the composition and activity of the photosynthetic apparatus under energy limitation, maximal specific acetylene reduction and hydrogen evolution activities increased by factors of 3 and 8, respectively, when illumination of the culture was raised from 3,000 to about 15,000 lx. Above 15,000 lx, both activities of nitrogenase approached constancy.We, therefore, conclude that neither under energy limitation nor under nitrogen limitation the function of nitrogenase depended on the photosynthetic activities. Moreover, it is suggested that light did not influence nitrogenase activity under conditions of nitrogen limitation, while under conditions of energy limitation light seemed to influence nitrogenase activities indirectly via glutamate consumption of the cells.     

Phosphorus inputs to Lake Naivasha,Kenya, from its catchment and the trophic state of the lake

The main river supplying Lake Naivasha, Kenya, the Malewa, drains a catchment given over to largely subsistence cultivation and animal husbandry. The lake itself is the focus for an intensive horticultural industry based upon irrigation from the lake. The Malewa, however, is relatively independent of the impact of industry, and so its contribution to eutrophication of the lake was evaluated. Two periods of study, a very wet-dry and a `normal' wet-dry season showed that the river contribution of phosphorus led to a total phosphorus loading of 1.4 g m<sup>–2</sup> lake surface ann<sup>–1</sup> in the very wet period compared to 0.2 in the `normal'. Chlorophyll `a' in the open water of the lake was significantly related to soluble reactive phosphorus. The lake is now eutrophic by normal limnological criteria.     

Preparation of polyclonal antibody specific for AtPLC4, an Arabidopsis phosphatidylinositol-specific phospholipase C in rabbits

Phosphoinositide-specific phospholipase Cs (PI-PLCs) are important enzymes in eukaryotes, which catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. The Arabidopsis genome contains nine putative PI-PLC genes. AtPLC4, an abiotic stress induced gene, has been reported to encode an active PI-PLC isoform. However, the exact roles of putative AtPLC4 in plant remain to be elicited. The first 108 amino acid residues of the N-terminal of AtPLC4, referred to as AtPLC4 N, was expressed as a recombinant protein in Escherichia coli and used as antigen in generating antibody. Purified recombinant proteins including AtPLC1 to AtPLC5, AtPLC8, AtPLC9 and AtPLC4 N were transferred onto the same blot to test specificity of the prepared antibody. Western blot result shows that only AtPLC4 and AtPLC4 N can be recognized by the antibody. The antibody recognized a protein of approximately 68kDa in the plasma membrane fraction and cytosolic fractions prepared from Arabidopsis thaliana plants. This corresponds very well with the calculated molecular weight of AtPLC4. The results suggest that AtPLC4 may encode a plasma membrane-associated protein.     

Seeing green bacteria in a new light: genomics-enabled studies of the photosynthetic apparatus in green sulfur bacteria and filamentous anoxygenic phototrophic bacteria

Based upon their photosynthetic nature and the presence of a unique light-harvesting antenna structure, the chlorosome, the photosynthetic green bacteria are defined as a distinctive group in the Bacteria. However, members of the two taxa that comprise this group, the green sulfur bacteria (Chlorobi) and the filamentous anoxygenic phototrophic bacteria (Chloroflexales), are otherwise quite different, both physiologically and phylogenetically. This review summarizes how genome sequence information facilitated studies of the biosynthesis and function of the photosynthetic apparatus and the oxidation of inorganic sulfur compounds in two model organisms that represent these taxa, Chlorobium tepidum and Chloroflexus aurantiacus. The genes involved in bacteriochlorophyll (BChl) c and carotenoid biosynthesis in these two organisms were identified by sequence homology with known BChl a and carotenoid biosynthesis enzymes, gene cluster analysis in Cfx. aurantiacus, and gene inactivation studies in Chl. tepidum. Based on these results, BChl a and BChl c biosynthesis is similar in the two organisms, whereas carotenoid biosynthesis differs significantly. In agreement with its facultative anaerobic nature, Cfx. aurantiacus in some cases apparently produces structurally different enzymes for heme and BChl biosynthesis, in which one enzyme functions under anoxic conditions and the other performs the same reaction under oxic conditions. The Chl. tepidum mutants produced with modified BChl c and carotenoid species also allow the functions of these pigments to be studied in vivo.     

The influence of variations in irradiance upon morphology in an unattached form of Gracilaria gracilis (Stackhouse) Steentoft during field cultivation, South Primorye, Russia

The changes in morphology and anatomy of an unattached form ofGracilaria gracilis from Southern Primorye, Russia, wereinvestigated as a response to different photon irradiances. Plants were grownat5, 10, 60 and 95% of the incident PAR in the field from mid July to midSeptember, and a quantitative evaluation of various morphologicalcharacteristics was made: thickness and length of branches, thickness of thecuticle and cell walls, cell length and diameter, the ratios between the areaofphotosynthetic and structural tissue. All characteristics showed variationsthatcould be attributed to irradiance. The narrow light range of 60–95% PARwas the growth optimum for Gracilaria gracilis. At theseirradiances, the biomass increase was related to the growth of the branches inlength (r = 0.95, P < 0.05) and thickness (r= 0.99, P < 0.05), and increased branch density.Growth of the branches only in length, the formation of the thin cortical cellwalls and cuticle, and increased ratios of photosynthetic to structural tissueswere among the effects of low irradiances (5–10% PAR) on plantmorphology.The relationship of morphology and productivity factors to irradiance intensityprovide evidence for their environmental rather than genetic control.Differences in thallus morphology and anatomy together with variations inlevelsof photosynthetic pigments are discussed in terms of the adaptive strategies ofunattached Gracilaria gracilis to light fluctuations.     

犬种布鲁氏菌ZG株单克隆抗体4H3抗原模拟表位的筛选及分析

【背景】目前犬布鲁氏菌病诊断存在一定的困难。【目的】筛选并研究犬种布鲁氏菌单克隆抗体4H3株的特异性抗原表位。【方法】利用噬菌体肽库展示技术,以犬种布鲁氏菌单克隆抗体4H3株作为靶分子,包被酶标板,用12肽随机肽库经过3轮生物淘洗程序进行筛选。经过3轮筛选后,噬菌体产出率从5.00×10^-7增加到9.84×10^-6,假阳性率逐轮降低。从第3轮筛选的阳性克隆中随机挑取14个进行增殖,提取基因组DNA,进行测序分析;并通过iELISA和cELISA检测阳性克隆的亲和性和特异性。【结果】14株阳性单克隆噬菌体共出现3种不同的短肽序列,分别是KMSIRHPIRLPI、ILRRRRKRIIQI和QRIHMRLTTQS;iELISA结果表明3种短肽序列与单克隆抗体的亲和性依次为KMSIRHPIRLPI>ILRRRRKRIIQI>QRIHMRLTTQS;cELISA结果显示短肽KMSIRHPIRLPI和ILRRRRKRIIQI特异性较强。对亲和性较强、特异性较高的2条短肽KMSIRHPIRLPI和ILRRRRKRIIQI展开具体分析,比对分析表...     

Inhibitory effects of myxothiazol and 2-n-heptyl-4-hydroxyquinoline-N-oxide on the auxiliary electron transport pathways of Rhodobacter capsulatus

The effects of various electron transport inhibitors upon the rates of reduction NO ₃ ^- , dimethyl sulphoxide (DMSO) and N₂O in anaerobic suspensions of Rhodobacter capsulatus have been studied. A new method for the determination of the rates of reduction of these auxiliary oxidants in intact cells is presented, based on the proportionality observed between the concentration of oxidant and the duration of the electrochromic carotenoid bandshift. For NO ₃ ^- and N₂O good agreement was found between rates of reduction determined using electrodes and those determined by the electrochromic method.Myxothiazol and antimycin A had no effect on the rates of reduction of NO ₃ ^- and DMSO suggesting that the cytochrome b/c ₁complex is not involved in electron transport to these oxidants. 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) inhibited at two sites, one within the cytochrome b/c ₁complex and the other on the nitrate reducing pathay, but had no effect on electron transport to N₂O or DMSO. In both intact cells and cell free extracts, HOQNO had no effect on the nitrate dependent re-oxidation of reduced methylviologen (MVH₂), a direct electron donor to nitrate reductase.Our data are consistent with a branch point for the auxiliary electron transport pathways at the level of the ubiquinone pool.Non-standard abbreviations HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - TMAO trimethylamine-N-oxide - DMSO dimethyl-sulphoxide - membrane potential - MVH₂ reduced methyl viologen     

Consistent Sets of Spectrophotometric Chlorophyll Equations for Acetone, Methanol and Ethanol Solvents

A set of equations for determining chlorophyll a (Chl a) and accessory chlorophylls b, c ₂ , c ₁ + c ₂ and the special case of Acaryochloris marina, which uses Chl d as its primary photosynthetic pigment and also has Chl a, have been developed for 90% acetone, methanol and ethanol solvents. These equations for different solvents give chlorophyll assays that are consistent with each other. No algorithms for Chl c compounds (c ₂ , c ₁ + c ₂) in the presence of Chl a have previously been published for methanol or ethanol. The limits of detection (and inherent error, ± 95% confidence limit), for chlorophylls in all organisms tested, was generally less than 0.1 μg/ml. The Chl a and b algorithms for green algae and land plants have very small inherent errors (< 0.01 μg/ml). Chl a and d algorithms for Acaryochloris marina are consistent with each other, giving estimates of Chl d/a ratios which are consistent with previously published estimates using HPLC and a rarely used algorithm originally published for diethyl ether in 1955. The statistical error structure of chlorophyll algorithms is discussed. The relative error of measurements of chlorophylls increases hyperbolically in diluted chlorophyll extracts because the inherent errors of the chlorophyll algorithms are constants independent of the magnitude of absorbance readings. For safety reasons, efficient extraction of chlorophylls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of chlorophylls. The methanol algorithms would be convenient for assays associated with HPLC work.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Nutrient optima and tolerances of benthic invertebrates,the effects of taxonomic resolution and testing of selected metrics in lakes using an extensive European data base

A large dataset from 1,077 lakes in Finland, Ireland, Sweden and the United Kingdom was collated to analyse the relationship between nutrient status and occurrence of different taxa, as well as between total phosphorus or chlorophyll and commonly used macroinvertebrate metrics developed for river assessment. We found that most taxa were associated with mesotrophic conditions (sensu OECD). Species associated with oligotrophic status included Baetis rhodani, Gammarus lacustris and plecopteran larvae, a group commonly associated with low nutrient status also in rivers. Species tolerant of eutrophic conditions were the chironomid larvae (Chironomus plumosus and Cryptochironomus defectus); and two species of tubificids (Psammoryctides barbatus and Potamothrix hammoniensis). For a number of taxa the associations of benthic invertebrates with nutrient state reported in the literature were not supported by analysis of the REBECCA data. The analysis indicated a variable response of littoral macroinvertebrates to eutrophication pressure when using common metrics developed for macroinvertebrates in rivers. Several metrics showed significantly different responses in lakes with different alkalinity, justifying the use of alkalinity for typing water bodies. These significant responses suggest that benthic invertebrates may be a useful component for classification of ecological status in lakes. The low amount of variance explained by the regressions (<30%), however, suggests that further harmonisation of sampling methods, as well as statistically more robust assessment tools are needed to increase the comparability of datasets and to improve the precision in the dose–response relationships.     

Effect of alteration in the global body plan on the deployment of morphogenesis-related protein epitopes labeled by the monoclonal antibody 12G9 in Tetrahymena thermophila

We have employed monoclonal antibodies to reinvestigate the janus mutants of the ciliate Tetrahymena thermophila, which cause reversal of circumferential polarity on the dorsal surface of the cell. This reversal brings about frequent ectopic expression of ventral cortical landmarks, such as a "secondary" oral apparatus, on the dorsal surface. The principal antibody employed, FXXXIX-12G9, immunolabels both transient cortical structures not directly associated with basal bodies (the fission line and the postoral meridional filament) and more permanent structures (apical band and oral crescent) that are associated with basal bodies. 12G9-immunolabeling of janus cells has revealed additional phenotypes, including disorder of ciliary rows. Further, this labeling has shown that the postoral meridional filament is often expressed and the apical band is frequently interrupted on the mid-dorsal surface of janus cells irrespective of whether or not these cells express a "secondary" oral apparatus. Of the permanent structures revealed by 12G9 immunofluorescence, modifications of the oral crescent (OC) are associated with prior modifications in the development of basal body-containing structures in the secondary oral apparatus. The formation of the apical band (AB) is also commonly abnormal in janus cells; analysis of specific abnormalities shows that the AB depends both on its initiation at a specific site near the anterior basal body of apical basal body couplets and on the normal location of these couplets just posterior to the fission line. We also have uncovered an intriguing difference in the reactivity of apical-band filaments to the 12G9 antibody in the two non-allelic janus mutants (janA1 and janC2) that we have investigated. Taken together, our observations indicate that the formation of new cellular structures at division depends both upon pre-existing cytoskeletal structures and upon the positional information provided by large-scale cellular polarities.