Quantitative changes and synthesis of cyanoprotein in whole body and tissues during development of the bean bug,Riptortus clavatus

Changes in biliverdin-binding cyanoprotein content in whole body and tissue extracts during development of nymphal and adult (non-diapause) bean bugs, Riptortus clavatus were analyzed by rocket immunoelectrophoresis (RIE). RIE using anti-CPegg serum can be used to determine the content of CP-A (Cp-1, 2 and 3) and CP-B (CP-4) separately. During the nymphal stage CP content of whole body changes cyclically in each instar. In the first nymphal instar, CPegg is the main CP which disappears during the first-second instar ecdysis. In nymphal bugs from the 2nd to 4th instars only CP-B (CP-4) is detected, and at the beginning of each instar the CP content is very low but increases toward the next ecdysis, after which CP decreases and disappears very rapidly. In the 5th nymphal instar, CP-B is the major CP but CP-A (CP-1, 2 and 3) is also detected. These changes in whole body CP content of 5th instar nymphs are observed in both females and males. The content of total CPs in the 5th instar nymph reaches about 1000 μg in the whole insect. During nymphal-adult ecdysis, nymphal CPs decrease and disappear at day 2 after emergence. In female adults CP-A (CP-1 only) increases rapidly after day 4 of adult emergence, while no CP is detected in male adults. In females CPs were detected only in the fat body, hemolymph and ovary. In the mid-5th-instar nymphs, CPs (CP-A and B) are mainly distributed in the hemolymph. CPs in the Hemolymph decrease during nymphal-adult ecdysis, whereas they increase in the fat body. CPs disappear from both the hemolymph and fat body by 2 days after ecdysis. Subsequently in the adult stage only CP-A increases again in the fat body and ovary. By tracer experiments using [³⁵S]-methionine, the fat body was shown to be the site of CP synthesis. CP-A and B synthetic activity was detected in nymphal females whereas, only CP-A synthesis was observed in adult females, while no CP synthesis was seen in adult males.     

Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae

Green fluorescent protein (GFP)-like pigments are responsible for the vivid colouration of many reef-building corals and have been proposed to act as photoprotectants. Their role remains controversial because the functional mechanism has not been elucidated. We provide direct evidence to support a photoprotective role of the non-fluorescent chromoproteins (CPs) that form a biochemically and photophysically distinct group of GFP-like proteins. Based on observations of Acropora nobilis from the Great Barrier Reef, we explored the photoprotective role of CPs by analysing five coral species under controlled conditions. In vitro and in hospite analyses of chlorophyll excitation demonstrate that screening by CPs leads to a reduction in chlorophyll excitation corresponding to the spectral properties of the specific CPs present in the coral tissues. Between 562 and 586 nm, the CPs maximal absorption range, there was an up to 50 % reduction of chlorophyll excitation. The screening was consistent for established and regenerating tissue and amongst symbiont clades A, C and D. Moreover, among two differently pigmented morphs of Acropora valida grown under identical light conditions and hosting subclade type C3 symbionts, high CP expression correlated with reduced photodamage under acute light stress.     

Amino acids in Tobamovirus coat protein controlling pepper L(1a) gene-mediated resistance

In pepper plants (genus Capsicum), the resistance against Tobamovirus spp. is conferred by L gene alleles. The recently identified L variant L^1a can recognize coat proteins (CPs) of Tobacco mild green mosaic virus Japanese strain (TMGMV‐J) and Paprika mild mottle virus Japanese strain (PaMMV‐J), but not of Pepper mild mottle virus (PMMoV), as the elicitor to induce resistance at 24 °C. Interestingly, L^1a gene‐mediated resistance against TMGMV‐J, but not PaMMV‐J, is retained at 30 °C. This observation led us to speculate that L^1a can discriminate between CPs of TMGMV‐J and PaMMV‐J. In this study, we aimed to determine the region(s) in CP by which L^1a distinguishes TMGMV‐J from PaMMV‐J. By using chimeric CPs consisting of TMGMV‐J and PaMMV‐J, we found that the chimeric TMGMV‐J CP, whose residues in the β‐sheet domain were replaced by those of PaMMV‐J, lost its ability to induce L^1a gene‐mediated resistance at 30 °C. In contrast, the chimeric PaMMV‐J CP with the β‐sheet domain replaced by TMGMV‐J CP was able to induce L^1a gene‐mediated resistance at 30 °C. Furthermore, viral particles were not detected in the leaves inoculated with either chimeric virus. These observations indicated that the amino acids within the β‐sheet domain were involved in both the induction of L^1a gene‐mediated resistance and virion formation. Further analyses using chimeric CPs of TMGMV‐J and PMMoV indicated that amino acids within the β‐sheet domain alone were not sufficient for the induction of L^1a gene‐mediated resistance by TMGMV‐J CP. These results suggest that multiple regions in Tobamovirus CP are implicated in the induction of L^1a gene‐mediated resistance.     

Space-Dependent Formation of Central Pair Microtubules and Their Interactions with Radial Spokes

Cilia and flagella contain nine outer doublet microtubules and a pair of central microtubules. The central pair of microtubules (CP) is important for cilia/flagella beating, as clearly shown by primary ciliary dyskinesia resulting from the loss of the CP. The CP is thought to regulate axonemal dyneins through interaction with radial spokes (RSs). However, the nature of the CP-RS interaction is poorly understood. Here we examine the appearance of CPs in the axonemes of a Chlamydomonas mutant, bld12, which produces axonemes with 8 to 11 outer-doublets. Most of its 8-doublet axonemes lack CPs. However, in the double mutant of bld12 and pf14, a mutant lacking the RS, most 8-doublet axonemes contain the CP. Thus formation of the CP apparently depends on the internal space limited by the outer doublets and RSs. In 10- or 11-doublet axonemes, only 3–5 RSs are attached to the CP and the doublet arrangement is distorted most likely because the RSs attached to the CP pull the outer doublets toward the axonemal center. The CP orientation in the axonemes varies in double mutants formed between bld12 and mutants lacking particular CP projections. The mutant bld12 thus provides the first direct and visual information about the CP-RS interaction, as well as about the mechanism of CP formation.     

The biosynthesis of Caryophyllaceae-like cyclic peptides in Saponaria vaccaria L. from DNA-encoded precursors

Cyclic peptides (CPs) are produced in a very wide range of taxa. Their biosynthesis generally involves either non‐ribosomal peptide synthases or ribosome‐dependent production of precursor peptides. Plants within the Caryophyllaceae and certain other families produce CPs which generally consist of 5–9 proteinogenic amino acids. The biological roles for these CPs in the plant are not very clear, but many of them have activity in mammalian systems. There is currently very little known about the biosynthesis of CPs in the Caryophyllaceae. A collection of expressed sequence tags from developing seeds of Saponaria vaccaria was investigated for information about CP biosynthesis. This revealed genes that appeared to encode CP precursors which are subsequently cyclized to mature CPs. This was tested and confirmed by the expression of a cDNA encoding a putative precursor of the CP segetalin A in transformed S. vaccaria roots. Similarly, extracts of developing S. vaccaria seeds were shown to catalyze the production of segetalin A from the same putative (synthetic) precursor. Moreover, the presence in S. vaccaria seeds of two segetalins, J [cyclo(FGTHGLPAP)] and K [cyclo(GRVKA)], which was predicted by sequence analysis, was confirmed by liquid chromatography/mass spectrometry. Sequence analysis also predicts the presence of similar CP precursor genes in Dianthus caryophyllus and Citrus spp. The data support the ribosome‐dependent biosynthesis of Caryophyllaceae‐like CPs in the Caryophyllaceae and Rutaceae.     

A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10⁷ M⁻¹. The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.     

The recombinant prepro region of TvCP4 is an inhibitor of cathepsin L-like cysteine proteinases of Trichomonas vaginalis that inhibits trichomonal haemolysis

Trichomonas vaginalis expresses multiple proteinases, mainly of the cysteine type (CPs). A cathepsin L-like 34 kDa CP, designated TvCP4, is synthesized as a 305-amino-acid precursor protein. TvCP4 contains the prepro fragment and the catalytic triad that is typical of the papain-like CP family of clan CA. The aim of this work was to determine the function of the recombinant TvCP4 prepro region (ppTvCP4r) as a specific inhibitor of CPs. We cloned, expressed, and purified the recombinant TvCP4 prepro region. The conformation of the purified and refolded ppTvCP4r polypeptide was verified by circular dichroism spectroscopy and fluorescence emission spectra. The inhibitory effect of ppTvCP4r was tested on protease-resistant extracts from T. vaginalis using fluorogenic substrates for cathepsin L and legumain CPs. In 1-D zymograms, the inhibitory effect of ppTvCP4r on trichomonad CP proteolytic activity was observed in the ∼97, 65, 39, and 30 kDa regions. By using 2-D zymograms and mass spectrometry, several of the CPs inhibited by ppTvCP4r were identified. A clear reduction in the proteolytic activity of several cathepsin L-like protein spots (TvCP2, TvCP4, TvCP4-like, and TvCP39) was observed compared with the control zymogram. Moreover, pretreatment of live parasites with ppTvCP4r inhibited trichomonal haemolysis in a concentration dependent manner. These results confirm that the recombinant ppTvCP4 is a specific inhibitor of the proteolytic activity of cathepsin L-like T. vaginalis CPs that is useful for inhibiting virulence properties depending on clan CA papain-like CPs.     

Blend miscibility of cellulose propionate with poly(N-vinyl pyrrolidone-co-methyl methacrylate)

The blend miscibility of cellulose propionate (CP) with poly(N-vinyl pyrrolidone-co-methyl methacrylate) (P(VP-co-MMA)) was investigated. The degree of substitution (DS) of CP used ranged from 1.6 to >2.9, and samples for the vinyl polymer component were prepared in a full range of VP:MMA compositions. Through DSC analysis and solid-state ¹³C NMR and FT-IR measurements, we revealed that CPs of DS < 2.7 were miscible with P(VP-co-MMA)s of VP ≥ ∼10 mol% on a scale within a few nanometers, in virtue of hydrogen-bonding interactions between CP-hydroxyls and VP-carbonyls. When the DS of CP exceeded 2.7, the miscibility was restricted to the polymer pairs using P(VP-co-MMA)s of VP = ca. 10–40 mol%; the scale of mixing in the blends concerned was somewhat larger (ca. 5–20 nm), however. The appearance of such a “miscibility window” was interpretable as an effect of intramolecular repulsion in the copolymer component. Results of DMA and birefringence measurements indicated that the miscible blending of CP with the vinyl polymer invited synergistic improvements in thermomechanical and optical properties of the respective constituent polymers. Additionally, it was found that the VP:MMA composition range corresponding to the miscibility window was expanded by modification of the CP component into cellulose acetate propionate.     

Purification and characterization of biliverdin-associated cyanoprotein from eggs and hemolymph of the bean bug,Riptortus clavatus (Heteroptera: Alydidae)

Biliverdin binding protein, cyanoprotein (CP), was purified from egg extracts of the bean bug, Riptortus clavatus. The preparation was shown to be homogeneous by DEAE-chromatography and polyacrylamide gel electrophoresis (PAGE). The native molecular weight (MW) of egg CP (CP_egg) was estimated to be 320,000 by PAGE and 335,000 by gel filtration. The apoprotein consisted of identical subunits with MW of 76,000. CP_egg had a high content of aromatic amino acids (17% mol ratio) with absorbance peaks at 370 and 620 nm which are characteristic of biliverdins. Native CP_egg was associated non-covalently with 16 molecules of biliverdin. CP_egg contained about 13.0% neutral sugar (mainly mannose) associated covalently with the apoprotein, no lipid was detected. In the hemolymph of the bugs, four CP bands (CP1, 2, 3 and 4) were detected by native PAGE. These bands were immunologically related and showed properties similar to CP_egg in biochemical analysis. Only CP1 was identical to CP_egg. CPs specific to the hemolymph, CP2, 3 and 4, were different from CP_egg in mobility by PAGE, pI and peptide mapping profiles.     

Changes in sulfate-reducing bacterial populations during the onset of black band disease

Factors that facilitate the onset of black band disease (BBD) of corals remain elusive, though anoxic conditions under the complex microbial mat and production of sulfide are implicated in necrosis of underlying coral tissues. This study investigated the diversity and quantitative shifts of sulfate-reducing bacterial (SRB) populations during the onset of BBD using real-time PCR (RT-PCR) and cloning approaches targeting the dissimilatory (bi)sulfite reductase (dsrA) gene. A quantitativePCR (qPCR) assay targeting the 16S rRNA gene also provided an estimate of total bacteria, and allowed the relative percentage of SRB within the lesions to be determined. Three Montipora sp. coral colonies identified with lesions previously termed cyanobacterial patches (CPs) (comprising microbial communities unlike those of BBD lesions), were tagged and followed through time as CP developed into BBD. The dsrA-targeted qPCR detected few copies of the gene in the CP samples (<65 per ng DNA), though copy numbers increased in BBD lesions (>2500 per ng DNA). SRB in CP samples were less than 1% of the bacterial population, though represented up to 7.5% of the BBD population. Clone libraries also demonstrated a shift in the dominant dsrA sequences as lesions shifted from CP into BBD. Results from this study confirm that SRB increase during the onset of BBD, likely increasing sulfide concentrations at the base of the microbial mat and facilitating the pathogenesis of BBD.     

Cytobiological studies on hemocyanin metabolism in the branchial heart complex of the common cuttlefish Sepia officinalis (Cephalopoda, Dibranchiata)

The present study confirms previous investigations that demonstrated a high copper content in the branchial heart and its appendage, and that gave the first indication that this organ complex might be involved in hemocyanin metabolism in Sepia officinalis L. Immunocytochemical localization of hemocyanin molecules within the endocytotic lysosomal system of the ovoid cells and tracer experiments with ¹²⁵I-labeled Sepia hemocyanin suggest its endocytotic uptake. Energy-dispersive X-ray microanalysis and histochemical methods reveal a high copper content within the ovoid cells of the branchial heart. In view of the turnover of the respiratory pigment in the branchial heart of Sepia officinalis L., we believe that the ovoid cells are a site of hemocyanin catabolism.     

Anaerobic/aerobic conditions and biostimulation for enhanced chlorophenols degradation in biocathode microbial fuel cells

Anaerobic/aerobic conditions affected bacterial community composition and the subsequent chlorophenols (CPs) degradation in biocathode microbial fuel cells (MFCs). Bacterial communities acclimated with either 4-chlorophenol (4-CP) or 2,4-dichlorophenol (2,4-DCP) under anaerobiosis can degrade the respective substrates more efficiently than the facultative aerobic bacterial communities. The anaerobic bacterial communities well developed with 2,4-DCP were then adapted to 2,4,6-trichlorophenol (2,4,6-TCP) and successfully stimulated for enhanced 2,4,6-TCP degradation and power generation. A 2,4,6-TCP degradation rate of 0.10 mol/m³/d and a maximum power density of 2.6 W/m³ (11.7 A/m³) were achieved, 138 and 13 % improvements, respectively compared to the controls with no stimulation. Bacterial communities developed with the specific CPs under anaerobic/aerobic conditions as well as the stimulated biofilm shared some dominant genera and also exhibited great differences. These results provide the most convincing evidence to date that anaerobic/aerobic conditions affected CPs degradation with power generation from the biocathode systems, and using deliberate substrates can stimulate the microbial consortia and be potentially feasible for the selection of an appropriate microbial community for the target substrate (e.g. 2,4,6-TCP) degradation in the biocathode MFCs.     

The conserved aromatic residue W122 is a determinant of potyviral coat protein stability,replication, and cell-to-cell movement in plants

Coat proteins (CPs) play critical roles in potyvirus cell-to-cell movement. However, the underlying mechanism controlling them remains unclear. Here, we show that substitutions of alanine, glutamic acid, or lysine for the conserved residue tryptophan at position 122 (W¹²²) in tobacco vein banding mosaic virus (TVBMV) CP abolished virus cell-to-cell movement in Nicotiana benthamiana plants. In agroinfiltrated N. benthamiana leaf patches, both the CP and RNA accumulation levels of three W¹²² mutant viruses were significantly reduced compared with those of wild-type TVBMV, and CP accumulated to a low level similar to that of a replication-deficient mutant. The results of polyprotein transient expression experiments indicated that CP instability was responsible for the significantly low CP accumulation levels of the three W¹²² mutant viruses. The substitution of W¹²² did not affect CP plasmodesmata localization or virus particle formation; however, the substitution significantly reduced the number of virus particles. The wild-type TVBMV CP could complement the reduced replication and abolished cell-to-cell movement of the mutant viruses. When the codon for W¹²² was mutated to that for a different aromatic residue, phenylalanine or tyrosine, the resultant mutant viruses moved systemically and accumulated up to 80% of the wild-type TVBMV level. Similar results were obtained for the corresponding amino acids of W¹²² in the watermelon mosaic virus and potato virus Y CPs. Therefore, we conclude that the aromatic ring in W¹²² in the core domain of the potyviral CP is critical for cell-to-cell movement through the effects on CP stability and viral replication.     

Predicting and managing the effects of hypersalinity on the fish community in solar salt fields in north-western Australia

Five concentrator ponds (CPs) of a solar salt field in Port Hedland, Western Australia were sampled by seine and gill nets over a 12‐month period in order to describe the fish community and examine relationships between diversity, abundance and catch per unit effort (CPUE) with salinity. Salinity varied between 40.2 and 113.7‰ during the sampling period. Forty‐one species of fishes were recorded from the CPs, with fewer species recorded from CPs of higher salinity. A significant inverse relationship was identified between salinity and the number of species (diversity) captured in gill nets, indicating that one species is lost with every 16‰ increase in salinity. A significant relationship between salinity and CPUE was also identified with gill‐net samples, indicating a reduction of 1 kg h^?1 with every increase in salinity of 5.5‰. As CPs are connected by one‐way flaps, fish movements are only possible into CPs of higher salinity. Thus, reductions in diversity, abundance and CPUE suggested fish mortalities, likely as a result of maximum or rapidly changing salinities exceeding the tolerance ability of individual species. As fish kills are not infrequent events in solar salt fields and result in economic losses due to loss of production and clean‐up costs, the results may allow managers to identify high risk species and times of year of fish kills by using salinity measurements. Commercial, indigenous and/or recreational fishing opportunities are viable options for reducing fish biomasses within the CPs and are discussed. Although absolute salinity values were higher than those recorded from tropical Australian estuaries, salinity deviations within each CP are similar to other estuaries and the effect on the ichthyo‐community is likely to be similar.     

The effect of (o-phenanthroline)2-cupric sulfate on several properties associated with (N-A+ + K+)-dependent adenosine triphosphatase.

Previous studies have shown that the large polypeptide of purified (Na⁺ + K⁺)-dependent adenosine triphosphatase (NaK ATPase) reacts to form a dimer and other higher oligomeric structures of the enzyme as a result of cross-linking with (o-phenanthroline)₂-cupric sulfate (CP). In the present communication, I show that both NaK ATPase activity and p-nitrophenylphosphatase (NPPase) activity decline rapidly and nearly in parallel when the enzyme is reacted with CP. Similarly, ATP binding is lost with kinetics close to those of ATPase activity and NPPase activity. The loss of ATPase activity, NPPase activity, and ATP binding occurs at a considerably faster rate than cross-linking of the large polypeptide, suggesting that CP may also be forming intrachain disulfide bonds. The binding of ouabain to NaK ATPase is also altered as a result of reacting the enzyme with CP. In marked contrast to ATP binding, however, ouabain binding is lost at a slower rate which closely parallels the rate of reaction of the large polypeptide to form cross-linked oligomeric structures.     

Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).     

Conjugated polymers for enhanced bioimaging

Background

Conjugated polymers (CPs) have been used for creating bioimaging tools or biosensors that provide a direct link between spectral signal and different biological processes. The detection schemes of these sensors are mainly employing the efficient light harvesting properties or the conformation sensitive optical properties of the CPs. Hence, the presence of biomolecules or biological events can be detected through fluorescence resonance energy transfer (FRET) between the CP and an acceptor molecule, or through their impact on the conformation of the conjugated backbone, which is seen as an alteration of the optical properties of the CP.

Scope of the review

In this review, the utilization of CPs for sensitive detection of DNA and protein conformational changes will be presented. The main part will be focused on the specific binding of CPs to protein deposits associated with protein misfolding diseases, such as Alzheimer's disease (AD), and the discovery that tailor-made CPs can be used for in vivo optical imaging of protein aggregates will be discussed.

Major conclusions

The unique optical properties of CPs can be used as molecular tools for sensitive detection of genetic material and for characterization of the pathological hallmarks associated with protein misfolding disorders, such as AD.

General significance

CPs are novel molecular tools that can be used for sensitive bioimaging of biological processes and these tools offer the possibility to study biological events in a complementary fashion to conventional techniques.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.