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.     

Mutant Native Outer Membrane Vesicles Combined with a Serogroup A Polysaccharide Conjugate Vaccine for Prevention of Meningococcal Epidemics in Africa

Background

The meningococcal serogroup A (MenA) polysaccharide conjugate vaccine used in Sub-Saharan Africa does not prevent disease caused by MenW or MenX strains, which also cause epidemics in the region. We investigated the vaccine-potential of native outer membrane vesicles with over-expressed factor H-binding protein (NOMV-fHbp), which targeted antigens in African meningococcal strains, and was combined with a MenA polysaccharide conjugate vaccine.

Methodology/Principal Findings

The NOMV-fHbp vaccine was prepared from a mutant African MenW strain with PorA P1.5,2, attenuated endotoxin (ΔLpxL1), deleted capsular genes, and over-expressed fHbp in variant group 1. The NOMV-fHbp was adsorbed with Al(OH)₃ and used to reconstitute a lyophilized MenA conjugate vaccine, which normally is reconstituted with liquid MenC, Y and W conjugates in a meningococcal quadrivalent conjugate vaccine (MCV4-CRM, Novartis). Mice immunized with the NOMV-fHbp vaccine alone developed serum bactericidal (human complement) activity against 13 of 15 African MenA strains tested; 10 of 10 African MenX strains, 7 of 7 African MenW strains, and 6 of 6 genetically diverse MenB strains with fHbp variant group 1 (including 1 strain from The Gambia). The combination NOMV-fHbp/MenA conjugate vaccine elicited high serum bactericidal titers against the two MenA strains tested that were resistant to bactericidal antibodies elicited by the NOMV-fHbp alone; the combination elicited higher titers against the MenA and MenW strains than those elicited by a control MCV4-CRM vaccine (P<0.05); and high titers against MenX and MenB strains. For most strains, the titers elicited by a control NOMV-fHbp knock out vaccine were <1∶10 except when the strain PorA matched the vaccine (titers >1∶000).

Conclusion/Significance

The NOMV-fHbp/MenA conjugate vaccine provided similar or higher coverage against MenA and MenW strains than a quadrivalent meningococcal conjugate vaccine, and extended protection against MenX strains responsible for epidemics in Africa, and MenB strains with fHbp in variant group 1.     

Effectiveness of Meningococcal C Conjugate Vaccine in Salvador,Brazil: A Case-Control Study

Background

During a citywide epidemic of serogroup C meningococcal disease in Salvador in 2010, Brazil, the state government initiated mass vaccination targeting two age groups with high attack rates: individuals aged <5 years and 10–24 years. More than 600,000 doses of meningococcal serogroup C conjugate vaccines were administered. We performed a case-control study to evaluate vaccine uptake, document vaccine effectiveness and identify reasons for non-vaccination.

Methods and Findings

Population-based surveillance identified patients with laboratory-confirmed invasive meningococcal C (MenC) disease during 2010. Information on MenC vaccination was obtained from case patients and age-matched individuals from the same neighborhoods. MenC vaccine effectiveness was estimated based on the exact odds ratios obtained by conditional logistic regression analysis. Of 51 laboratory-confirmed cases of serogroup C meningococcal disease among patients <5 and 10–24 years of age 50 were included in the study and matched with 240 controls. Overall case-fatality was 25%. MenC vaccine coverage among controls increased from 7.1% to 70.2% after initiation of the vaccination campaign. None of the 50 case patients but 70 (29.2%) of the 240 control individuals, including 59 (70.2%) of 84 matched with cases from the period after MenC vaccination, had received at least one MenC vaccine dose. Overall effectiveness of MenC was 98% with a lower 95% exact confidence limit of 89%.

Conclusions

MenC vaccines administered during the meningococcal epidemic were highly effective, suggesting that rapid vaccine uptake through campaigns contributed to control of meningococcal disease.     

O-Acetylation status of the capsular polysaccharides of serogroup Y and W135 meningococci isolated in the UK

At a time when tetravalent conjugate vaccines for meningococcal serogroups A/C/Y/W135 are being formulated the O-acetylation status of their respective capsular polysaccharides has not previously been studied in the UK for all components. Although this has been elucidated for serogroup C, little is known about the O-acetylation status of serogroups W135 and Y. Meningococcal serogroup W135 (n=181) and Y (n=90) isolates submitted to the PHLS Meningococcal Reference Unit in 1996, 2000 and 2001 were investigated for O-acetylation capsular status by dot blot assay. Eight per cent of W135 and 79% of Y isolates respectively were found to be O-acetylated with a similar distribution found in both carrier and case isolates. An increase in O-acetylated W135 isolates was noted between 2000 (0%) and 2001 (21%) which was not due to the introduction of the Hajj associated W135 (ET 37 complex; serosubtype P1.5,2) isolates, all of which were de-O-acetylated. Although the biological relevance of O-acetylation status is unknown for these serogroups, an understanding of O-acetylation status of the respective polysaccharides may provide useful insights into the optimal vaccine formulation.     

Serogroup quantitation of multivalent polysaccharide and polysaccharide-conjugate meningococcal vaccines from China

The active components of most meningococcal vaccines are four antigenic serogroup capsular polysaccharides (A, C, Y, W135). The vaccines, monovalent or multivalent mixtures of either free polysaccharides or polysaccharides conjugated to antigenic carrier proteins, may be in liquid or lyophilised formulations, with or without excipients. Acid hydrolysis and chromatographic methods for serogroup quantitation, which were previously optimised and qualified using polysaccharide-based standards and a narrow range of real vaccines, are here challenged with multiple lots of a broad assortment of additional multivalent polysaccharide-based meningococcal vaccine products. Centrifugal filtration successfully removed all interfering lactose excipient without loss of polysaccharides to allow for the determination of Y and W135 serogroups. Replicate operations by three different analysts indicated high method reproducibility. Results indicated some lot-to-lot and product-to-product variations. However, all vaccines were within general specifications for each serogroup polysaccharide, with the exception of all lots of one polysaccharide vaccine – which by these methods were found to be deficient in the serogroup A component only. These robust techniques are very useful for the evaluation of antigen content and consistency of manufacture. The deformulation, hydrolysis and chromatographic methods may be adaptable for the evaluation of other types of polysaccharide-based vaccines.     

Meningococcal polysaccharide vaccines: A review

Polysaccharides produced by Neisseria meningitidis are pharmaceutically important molecules, and are the active components of vaccines against N. meningitidis serogroups A, C, W135 and Y. Effective vaccines based on capsular polysaccharide, polysaccharide conjugates and outer membrane vesicles have been developed for strains expressing capsular polysaccharides that define the sero groups A, C, Y and W135. However, conventional approaches to develop a vaccine for group B strains have been largely unsuccessful. This review focuses on the various aspects of fermentative production of meningococcal polysaccharide from N. meningitidis, methods of conjugation for improving the immunogenicity of polysaccharide vaccine, and efficient and cost effective methods for the purification of N. meningitidis capsular polysaccharide and outer membrane vesicles. In addition, different analytical techniques for the quantitative determination of polysaccharide vaccine and evaluation of structural integrity of conjugate vaccine have been described.     

Prevalence and Phase Variable Expression Status of Two Autotransporters,NalP and MspA,in Carriage and Disease Isolates of Neisseria meningitidis

Neisseria meningitidis is a human nasopharyngeal commensal capable of causing life-threatening septicemia and meningitis. Many meningococcal surface structures, including the autotransporter proteins NalP and MspA, are subject to phase variation (PV) due to the presence of homopolymeric tracts within their coding sequences. The functions of MspA are unknown. NalP proteolytically cleaves several surface-located virulence factors including the 4CMenB antigen NhbA. Therefore, NalP is a phase-variable regulator of the meningococcal outer membrane and secretome whose expression may reduce isolate susceptibility to 4CMenB-induced immune responses. To improve our understanding of the contributions of MspA and NalP to meningococcal-host interactions, their distribution and phase-variable expression status was studied in epidemiologically relevant samples, including 127 carriage and 514 invasive isolates representative of multiple clonal complexes and serogroups. Prevalence estimates of >98% and >88% were obtained for mspA and nalP, respectively, with no significant differences in their frequencies in disease versus carriage isolates. 16% of serogroup B (MenB) invasive isolates, predominately from clonal complexes ST-269 and ST-461, lacked nalP. Deletion of nalP often resulted from recombination events between flanking repetitive elements. PolyC tract lengths ranged from 6–15 bp in nalP and 6–14 bp in mspA. In an examination of PV status, 58.8% of carriage, and 40.1% of invasive nalP-positive MenB isolates were nalP phase ON. The frequency of this phenotype was not significantly different in serogroup Y (MenY) carriage strains, but was significantly higher in invasive MenY strains (86.3%; p<0.0001). Approximately 90% of MenB carriage and invasive isolates were mspA phase ON; significantly more than MenY carriage (32.7%) or invasive (13.7%) isolates. This differential expression resulted from different mode mspA tract lengths between the serogroups. Our data indicates a differential requirement for NalP and MspA expression in MenB and MenY strains and is a step towards understanding the contributions of phase-variable loci to meningococcal biology.     

Determination of the linkages in some methylated,sialic acid-containing,meningococcal polysaccharides by mass spectrometry

The application of gas-liquid chromatography-mass spectrometric (g.l.c.-m.s.) analysis to a number of sialic acid-containing polysaccharides of meningococcal origin has been studied. Methylation of these polysaccharides by the Hakomori conditions resulted in both O- and N-methylation. Methanolysis of the methylated polysaccharides from serogroup C [(2→9)-linked], colominic acid [(2→8)-linked], and serogroups Y and W-135 [both (1→4)-linked], yielded the respective 4,7,8,4,7,9-, and 7,8,9-tri-O-methyl derivatives of methyl N-acetyl-N-methyl-β-D-neuraminate methyl glycoside. As model compounds, methyl N-acetyl-4,7,8,9-tetra-O-methyl-α-D-neuraminate methyl glycoside and its N-methyl derivative were also synthesized. All of the methylated derivatives could be identified on the basis of their typical fragmentation-patterns, indicating that this method is applicable to the determination of the position of linkages to sialic acid residues in biopolymers.     

The quantitative immunochemical determination of pneumococcal and meningococcal capsular polysaccharides by light scattering rate nephelometry

A quantitative nephelometric method was used for the measurement of the individual pneumococcal, as well as meningococcal, polysaccharides in the polyvalent vaccine final containers. This method is simple, rapid, inexpensive, and provides both qualitative and quantitative analyses of the polyvalent polysaccharide vaccines. By this method the individual pneumococcal types, 1, 2, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18C, 19F, 23F and 25 polysaccharides, were found to be present at 90-114% of the manufacturer's indicated concentrations; meningococcal group A, C, Y and W135 polysaccharides were at 90-108% of the manufacturer's listed concentrations. This nephelometric method coupled with gel filtration can also be used for measurement of the molecular sizes or stability of individual polysaccharides in the final container. Pneumococcal polysaccharide types 3, 6A, 9N and 19F, used as representative types, were treated with 0.5 N hydrochloric acid. The molecular sizes for types 3 and 9 N polysaccharides were stable to acid treatment. In contrast, types 6A and 19F polysaccharides were degraded. Heating meningococcal groups A, C, Y and W135 polysaccharides at 37 degrees C for 48 h did not affect their molecular size in the polyvalent vaccine.     

A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): Purification,primary structure,and inhibitory properties

Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K_a = 1.1 × 10⁹ M^?1 and 2.5 × 10⁹ M^?1, respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K_a = 4.5 × 10⁸ M^?1 and 1.2 × 10¹⁰ M^?1). Weak interaction with human plasmin (K_a = 1.2 × 10⁷ M^?1) was also revealed.     

Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies

Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS–mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which ranged from ∼ 3–50 kDa. Four groups were specific to V. vulnificus without cross reactivity to either other Vibrio spp. or other bacterial species. In dot blot based assays, one group of MAbs were specific to VVC, with a sensitivity of ∼ 1.6 × 10⁷ CFU ml^− 1 (∼ 1.6 × 10⁴ cells spot^− 1), and bound to proteins of ∼ 50 and ∼ 39 kDa. Other MAbs, binding to proteins ranging from ∼ 3–14 and ∼ 40 kDa, detected VVB (but not VVC) with high sensitivity at ∼ 1.6 × 10⁵ and 4 × 10⁶ CFU ml^− 1 (∼ 1.6 × 10² and 4 × 10³ cells spot^− 1), respectively. In addition, certain MAbs were able to recognize V. vulnificus in tissues by means of immunohistochemistry. The remaining groups demonstrated cross reactivity to Vibrio fluvialis. MAbs from this study can, therefore, detect the difference between some isolates of V. vulnificus and in addition to pathogen detection may, with further antibodies, form the basis of serovar typing isolates in the future.     

2,5-Diaryloxadiazoles and their precursors as novel inhibitors of cathepsins B,H and L

High levels of cathepsins indicated in various pathological conditions like arthritis, cancer progressions, and atherosclerosis explains the need to explore potential inhibitors of these proteases which can be of great therapeutic significance. We, in the present work, report the synthesis of some 2,5-diaryloxadiazoles from N-subsitutedbenzylidenebenzohydrazides. The synthesized compounds were screened for their inhibitory potential on cathepsins B, H and L. Structure Activity Relationship studies show that 2,5-diaryloxadiazoles were less inhibitory than their precursors. 1i and 2k have been found to be most inhibitory to cathepsins B and L. Their K_i values have been calculated as 11.38 × 10⁻⁸ M and 66.4 × 10⁻⁸ M for cathepsin B and 4.2 × 10⁻⁹ M and 47.31 × 10⁻⁹ M for cathepsin L, respectively. However, cathepsin H activity was maximally inhibited by compounds, 1e and 2c with K_i values of 4.4 × 10⁻⁷ M and 5.6 × 10⁻⁷ M, respectively. Enzyme kinetic studies suggest that these compounds are competitive inhibitors to the enzymes. The results have been compared with docking results obtained using iGemDock.     

Molecular divergence of the sia locus in different serogroups of Neisseria meningitidis expressing polysialic acid capsules

The serogroups B, C, W135 and Y of Neisseria meningitidis express chemically and immunologically distinct capsular polysaccharides containing sialic acid. In the case of serogroup B meningococci sialic acid is synthesized by the gene products of a locus termed sia and forms the homopolymers of the capsule. The organization of the genes required for sialic acid synthesis in serogroups B, C, W135 and Y was elucidated by PCR technology. Cloning, sequencing and the functional expression of the polysialyltransferase (PST) genes of serogroups B and C demonstrated that the difference in capsule composition derives from the presence of related, but distinct siaD genes coding for PSTs. Analysis of meningococci of serogroups W135 and Y expressing sialic acid heteropolymers revealed that the DNA sequences of the corresponding genetic loci in these serogroups were highly homologous, but differed completely from the siaD genes of serogroups B and C. This finding suggests that enzymes unrelated to those of serogroups B and C are required for the formation of sialic acid heteropolymers characteristic of the capsules of serogroups W135 and Y.     

Cyclic pollen production in Cedrus deodara

Based on a seven-year study of pollen production and release in two different natural populations of Cedrus deodara from Garhwal Himalaya, India, we determined that pollen output follows a two-year cycle. Pollen productivity determinations considered various sources of variability, including the number of pollen strobili per branch, strobili per tree, microsporophylls per tree and pollen grains per tree. Each of these parameters revealed significant year-to-year and population effects. Microsporangium dehiscence took from 2.5 to 3.5 days. Maximum dehiscence was observed between 12:00 and 14:00 h, which coincides with diurnal highest temperature and lowest relative humidity. Annual production of pollen per tree varied from averages of 4.7 × 10⁹, 7.2 × 10⁹ and 5.1 × 10⁹ in years of low production, with alternate high years, producing 12.6 × 10⁹, 14.1 × 10⁹, 13.3 × 10⁹ and 14.0 × 10⁹ pollen grains per tree. Annual pollen production in individual trees of C. deodara ranged from 1.4 × 10⁹ to 22.3 × 10⁹.     

Pathogenicity of entomopathogenic fungus,Metarhizium anisopliae MET-GRA4 isolate on dengue vectors,Aedes albopictus and Aedes aegypti mosquito larvae (Diptera: Culicidae)

Considering the rapid transmission of the dengue virus, substantial efforts need to be conducted to ward-off the epidemics of dengue viruses. The control effort is depending on chemical insecticides and had aroused undesirable conflicts of insecticide resistance. Here, we study the entomopathogenic fungus, Metarhizium anisopliae as a promising new biological control agent for vector control. The pathogenicity effects of Metarhizium anisopliae against field and laboratory strains of Aedes albopictus and Aedes aegypti larvae were tested using the larvicidal bioassay technique. The results demonstrate that the treatments using M. anisopliae isolate MET-GRA4 were highly effective and able to kill 100% of both Ae. albopictus and Ae. aegypti mosquito larvae at a conidia concentration of 1 × 10?/ml within 7 days of the treatment period. The fungus displayed high larvicidal activity against laboratory and field strain of Ae. aegypti larvae with LC₅₀ values (9.6 × 10³/ml, 1.3 × 10³/ml) and LC₉₅ values (1.2 × 10?/ml, 5.5 × 10⁵/ml) respectively. For Ae. albopictus, LC₅₀ values for laboratory and field strains were (1.7 × 10⁴/ml, 2.7 × 10⁴/ml) and the LC₉₅ values were (2.1 × 10?/ml, 7.0 × 10⁵/ml) respectively. Interestingly, the susceptibility of field strain towards M. anisopliae was higher as compared to the laboratory strain Aedes larvae. In which, the causative agents of all the dead larvae were verified by the virulence of M. anisopliae and caused morphological deformities on larval body. The findings from this study identify this isolate could be an effective potential biocontrol agent for vector mosquitoes in Malaysia.     

Molecular divergence of the sia locus in different serogroups of Neisseria meningitidis expressing polysialic acid capsules

The serogroups B, C, W135 and Y of Neisseria meningitidis express chemically and immunologically distinct capsular polysaccharides containing sialic acid. In the case of serogroup B meningococci sialic acid is synthesized by the gene products of a locus termed sia and forms the homopolymers of the capsule. The organization of the genes required for sialic acid synthesis in serogroups B, C, W135 and Y was elucidated by PCR technology. Cloning, sequencing and the functional expression of the polysialyltransferase (PST) genes of serogroups B and C demonstrated that the difference in capsule composition derives from the presence of related, but distinct siaD genes coding for PSTs. Analysis of meningococci of serogroups W135 and Y expressing sialic acid heteropolymers revealed that the DNA sequences of the corresponding genetic loci in these serogroups were highly homologous, but differed completely from the siaD genes of serogroups B and C. This finding suggests that enzymes unrelated to those of serogroups B and C are required for the formation of sialic acid heteropolymers characteristic of the capsules of serogroups W135 and Y. Received: 24 June 1997 / Accepted: 23 September 1997     

Virulence of a South African isolate of the <Emphasis Type="Italic">Cryptophlebia leucotreta</Emphasis> granulovirus to <Emphasis Type="Italic">Thaumatotibia leucotreta</Emphasis> neonate larvae

Surface inoculation dose–response and time–response bioassays and detached fruit bioassays were conducted with a novel South African isolate of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) against Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Noctuidae) neonate larvae. LC₅₀ and LC₉₀ values were estimated to be 4.095 × 10³ and 1.185 × 10⁵ OBs ml⁻¹, respectively. LT₅₀ and LT₉₀ values were estimated to be 4 days 22 h and 7 days 8 h, respectively, categorising the virus as a fast or type 2 granulovirus. There was a conspicuous difference in behaviour between larvae on inoculated diet and untreated diet, resulting in a significant reduction in penetration of diet. Bioassays on detached Navel oranges revealed LC₅₀ and LC₉₀ values of 9.310 × 10⁷ and 1.515 × 10⁹ OBs ml⁻¹, when using data on numbers of larvae per fruit rather than on numbers of infested fruit. Field trials will be conducted.     

Detection Methanogens in Newly Settled Sediments from Xuanwu Lake in Nanjing, China

Sediments from Xuanwu Lake have been dredged in the past 3 years to improve the water quality, but methanogenesis should still exist in the newly settled sediment. Methane production, methanogens, and physiochemical parameters were detected in the surface sediments (0–5 cm) and/or vertical sediments (0–21 cm, segmented at interval of 3 cm). Methane flux at water–air interface varied among five detected sites. Principal component analysis showed that CH₄ flux, content of water and the concentration of total nitrogen (TN), CH₄ and organic matters (OM) weighed most heavily on the component I in surface sediments while different patterns were observed for vertical sediments. The copy number of the 16S rRNA gene for bacteria was lower in the surface sediment (0–6 cm) than that in deeper sediments (12–21 cm), while 16S rRNA genes of Archaea were almost evenly distributed in the vertical sediments. Representatives belonging to the orders Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in all samples of the vertical sediments, except that no members of the Methanococcales were detected in the samples at 0–6 cm. The level of Methanobacteriales reached a highest density at 18.1 × 10⁴ copies g⁻¹ dry weight (dw) at 6–9 cm; for Methanosarcinales (76.89 × 10⁶ copies g⁻¹ dw) and Methanococcales (82.70 × 10³ copies g⁻¹ dw) at 12–15 cm, whereas for Methanomicrobiales (43.37 × 10⁶ copies g⁻¹ dw) at 9–12 cm. Methanosarcinaceae and Methanosaetaceae reached to their highest densities at 6–9 and 9–12 cm, respectively. These data provided useful information for better understanding the methanogenesis in the newly settled sediments of a recently dredged lake.     

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.     

Flow‐injection chemiluminescence determination of ofloxacin using the Ru(bpy)2(CIP)2+−Ce(IV) system and its application

This paper reports a flow‐injection chemiluminescence method for the determination of ofloxacin (OFLX) using the Ru(bpy)₂(CIP)²⁺–Ce(IV) system. Under the optimum conditions, the relative CL intensity was proportional to the concentration of OFLX in the range 3.0 × 10^–8–1.0 × 10^–5 mol/L and the detection limit was 4.2 × 10^–9 mol/L. The proposed method has been successfully applied to the determination of ofloxacin in pharmaceuticals and human urine. The chemiluminescence mechanism of the system is also discussed. Copyright © 2008 John Wiley & Sons, Ltd.