Denying the Truth Does Not Change the Facts: A Systematic Analysis of Pseudoscientific Denial of Complex Regional Pain Syndrome.

PURPOSE: Several articles have claimed that complex regional pain syndrome (CRPS) does not exist. Although a minority view, it is important to understand the arguments presented in these articles. We conducted a systematic literature search to evaluate the methodological quality of articles that claim CRPS does not exist. We then examined and refuted the arguments supporting this claim using up-to-date scientific literature on CRPS. METHODS: A systematic search was conducted in MEDLINE, EMBASE and Cochrane CENTRAL databases. Inclusion criteria for articles were (a) a claim made that CRPS does not exist or that CRPS is not a distinct diagnostic entity and (b) support of these claims with subsequent argument(s). The methodological quality of articles was assessed if possible. RESULTS: Nine articles were included for analysis: 4 narrative reviews, 2 personal views, 1 letter, 1 editorial and 1 case report. Seven points of controversy were used in these articles to argue that CRPS does not exist: 1) disagreement with the label "CRPS"; 2) the "unclear" pathophysiology; 3) the validity of the diagnostic criteria; 4) CRPS as a normal consequence of immobilization; 5) the role of psychological factors; 6) other identifiable causes for CRPS symptoms; and 7) the methodological quality of CRPS research. CONCLUSION: The level of evidence for the claim that CRPS does not exist is very weak. Published accounts concluding that CRPS does not exist, in the absence of primary evidence to underpin them, can harm patients by encouraging dismissal of patients' signs and symptoms.

Complete Nucleotide Sequence of a Novel Hibiscus-Infecting Cilevirus from Florida and Its Relationship with Closely Associated Cileviruses.

The complete nucleotide sequence of a recently discovered Florida (FL) isolate of hibiscus-infecting cilevirus (HiCV) was determined by Sanger sequencing. The movement and coat protein gene sequences of the HiCV-FL isolate are more divergent than other genes of the previously sequenced HiCV-HI (Hawaii) isolate.

First Report of Citrus leprosis virus Nuclear Type in Sweet Orange in Colombia.

Colombia is ranked 18th in the world in citrus production and contributed 0.9% of the total world share. Among four important citrus-producing regions of Colombia, the Orinoco region (3 to 6°N, 68 to 74°W) consists of two citrus-producing states, Meta and Casanare. Citrus leprosis is the most important viral disease of citrus in Colombia (1,3). Three types of Citrus leprosis virus (CiLV) infect citrus, producing leprosis-like lesion symptoms. Two of the three CiLV species, Citrus leprosis virus cytoplasmic type (CiLV-C) and cytoplasmic type 2 (CiLV-C2), produce particles only in the cytoplasm (3). The other species, Citrus leprosis virus nuclear type (CiLV-N), produces particles in both the cytoplasm and nucleus (4). CiLV-C is more prevalent and destructive while CiLV-N has been reported only in Brazil, Panama, and Mexico (4). Interestingly, both CiLV-C and -C2 were reported from the same regions of Meta and Casanare States in Colombia in 2004 and 2012 (1,3). CiLV-C lesions are usually rounded (initially 2 to 3 mm in diameter and extending up to 30 mm), have dark-brown or greenish central chlorotic spots, and are surrounded by yellow halos. CiLV-N lesions have been described as smaller in size and form three well-defined regions including a necrotic center with an intermediate orange color halo and an outer chlorotic halo (2). In 2013, 'Valencia' sweet orange (Citrus sinensis L.) leaves with suspected CiLV-N symptoms were collected from 8 plants in Casanare State and shipped under permit to the USDA-APHIS-PPQ-CPHST, Beltsville, MD. Total RNA from symptomatic and healthy sweet orange leaves were extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). RT-PCR primers specific to CiLV-C, CiLV-C2 (3), and CiLV-N nucleocapsid (N) (CiLV-N-NPF: 5'-ATGGCTAACCCAAGTGAGATCGATTA-3'; CiLV-N-NPR: 5'-AGTTGCCTTGAGATCATCACATTGGT-3') and putative matrix protein (M) genes (CiLV-N-MF: 5'-ATGTCTAAACAGATTAATATGTGCACTGTG-3'; CiLV-N-MR: 5'-CTAACCACTGGGTCCCGC-3') were utilized to identify the CiLV associated with the leprosis-affected leaf samples from Casanare. RT-PCR with CiLV-C primers failed to produce any amplicon, but CiLV-N primers successfully amplified the partial N gene (681 bp) and entire M gene (552 nt) amplicons from multiple leaves of all leprosis samples. In addition, a 795-bp amplicon specific to CiLV-C2 also was amplified from the CiLV-N suspected samples. Similar results were obtained when the vector, flat spider mite (Brevipalpus spp.) total RNA was used as template for RT-PCR. For further confirmation, each amplicon was cloned and sequenced. Sequencing of the N and M gene amplicons of CiLV-N (accession nos. KJ195893 and KJ195894) and coat protein gene of CiLV-C2 showed 97 to 99% nucleotide sequence identity with the CiLV-N M2345 isolate sequence (KF209275) from Mexico (4) and CiLV-C2 L147V1 isolate sequence (JX000024) from Colombia (3), respectively. Phylogenetic analyses of these N and M protein gene sequences confirmed a mixed infection of the same plant with two viruses, one from an unassigned new genus Dichorhavirus (CiLV-N) and another from genus Cilevirus (CiLV-C2). This is the first report of CiLV-N in Colombia, and also the first report of an occurrence of CiLV-N in mixed infection with CiLV-C2. All three known species of CiLV occur in the Orinoco region of Colombia. References: (1) M. G. León et al. Plant Dis. 90: 682, 2006. (2) J. P. R. Marques et al. Anais da Academia Brasileira de Ciências 82:501, 2010. (3) A. Roy et al. Phytopathology 103:488, 2013. (4) A. Roy et al. Genome Announc. 1(4): e00519-13, 2013.

Development of Primers and Probes for Genus and Species Specific Detection of 'Candidatus Liberibacter Species' by Real-Time PCR.

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different 'Candidatus Liberibacter species', 'Ca. Liberibacter asiaticus', 'Ca. Liberibacter americanus', and 'Ca. Liberibacter africanus', which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of 'Ca. Liberibacter' at the genus level, providing increased convenience. Recent molecular analyses of 'Ca. Liberibacter species' and other bacteria suggest that the rpoB gene (encoding the ß-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus 'Ca. Liberibacter species', enabling detection of 'Ca. Liberibacter' at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected 'Ca. Liberibacter solanacearum', a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

Acquisition and Transmissibility of U.S. Soybean dwarf virus Isolates by the Soybean Aphid, Aphis glycines.

Soybean dwarf virus (SbDV) exists as several distinct strains based on symptomatology, vector specificity, and host range. Originally characterized Japanese isolates of SbDV were specifically transmitted by Aulacorthum solani. More recently, additional Japanese isolates and endemic U.S. isolates have been shown to be transmitted by several different aphid species. The soybean aphid, Aphis glycines, the only aphid that colonizes soybean, has been shown to be a very inefficient vector of some SbDV isolates from Japan and the United States. Transmission experiments have shown that the soybean aphid can transmit certain isolates of SbDV from soybean to soybean and clover species and from clover to clover and soybean with long acquisition and inoculation access periods. Although transmission of SbDV by the soybean aphid is very inefficient, the large soybean aphid populations that develop on soybean may have epidemiological potential to produce serious SbDV-induced yield losses.

Murraya paniculata and Related Species as Potential Hosts and Inoculum Reservoirs of 'Candidatus Liberibacter asiaticus', Causal Agent of Huanglongbing.

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of 'Candidatus Liberibacter asiaticus' from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted 'Ca. L. asiaticus' into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of 'Ca. L. asiaticus' from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of 'Ca. L. asiaticus' in infected Murraya.

Cultivation of 'Candidatus Liberibacter asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus' associated with huanglongbing.

A new medium designated Liber A has been designed and used to successfully cultivate all three 'Candidatus Liberibacter spp.,' the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of 'Ca. Liberibacter spp.' for four or five single-colony transfers before viability declined. Colonies, positive for 'Ca. L. asiaticus' by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex, and 0.1 to 0.3 mm after 3 to 4 days. Suspect 'Ca. L. asiaticus' and 'Ca. L. americanus' cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped, 0.3 to 0.4 by 0.5 to 2.0 microm, often with fimbriae-like appendages. Two strains of 'Ca. L. asiaticus' and one of 'Ca. L. americanus' grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of 'Ca. L. asiaticus' and 'Ca. L. americanus' associated with symptoms of HLB.

Prunus Host Range of Plum pox virus (PPV) in the United States by Aphid and Graft Inoculation.

Plum pox (Sharka) is a serious virus disease of stone fruits caused by the Plum pox virus (PPV). To determine which species could function as potential hosts and virus reservoirs, we used aphid transmission and bud or chip grafting to evaluate the susceptibility of commercial, ornamental, and wild Prunus species to isolates of PPV found in Pennsylvania, USA. Following inoculation, test trees were observed for symptoms, analyzed by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), back-assayed to healthy peach, and followed through at least four cold-induced dormancy (CID) cycles over 4 years. Thirty-one of 33 Prunus species and cultivars were systemically infected following aphid transmission. Systemic infection could not be detected in P. cerasus (sour cherry) and P. × 'Snofozam' (Snow Fountains) despite repeated aphid inoculation attempts. Following grafting of PPV-infected budwood, all 40 species and varieties became infected, although species differed in their susceptibility. Within most species, some individual plants remained PPV negative throughout the study despite repeated inoculations. Infection in some species could be detected only through quantitative reverse transcription (RT)-PCR. Most species displayed clear symptoms, were highly positive by ELISA and RT-PCR, and could be back-inoculated into peach seedlings following CID. Our results indicate that a wide range of native and ornamental Prunus species are susceptible to U.S. isolates of PPV-D.

Evidence for a hallucinogen dependence syndrome developing soon after onset of hallucinogen use during adolescence.

This study uses latent class methods and multiple regression to shed light on hypothesized hallucinogen dependence syndromes experienced by young people who have recently initiated hallucinogen use. It explores possible variation in risk. The study sample, identified within public-use data files of the 1999 National Household Survey on Drug Abuse (NHSDA), consists of 1186 recent-onset hallucinogen users, defined as having initiated hallucinogen use within 24 months of assessment (median elapsed time since onset of use -12 to 13 months). The recent-onset users in this sample were age 12 to 21 at the time of assessment and were between the ages of 10 and 21 at the time of their first hallucinogen use. The NHSDA included items to assess seven clinical features often associated with hallucinogen dependence, which were used in latent class modelling. Latent class analysis, in conjunction with prior theory, supports a three-class solution, with 2% of recent-onset users in a class that resembles a hallucinogen dependence syndrome, whereas 88% expressed few or no clinical features of dependence. The remaining 10% may reflect users who are at risk for dependence or in an early stage of dependence. Results from latent class regressions indicate that susceptibility to rapid transition from first hallucinogen use to onset of this hallucinogen dependence syndrome might be influenced by hallucinogenic compounds taken (for example, estimated relative risk, RR = 2.4, 95% CI = 1.6, 7.6 for users of MDMA versus users of LSD). Excess risk of rapid transition did not appear to depend upon age, sex, or race/ethnicity.

Structure-function analysis of the ADAM family of disintegrin-like and metalloproteinase-containing proteins (review).

The ADAMs belong to a disintegrin-like and metalloproteinase-containing protein family that are zinc-dependent metalloproteinases. These proteins share all or some of the following domain structure: a signal peptide, a propeptide, a metalloproteinase, a disintegrin, a cysteine-rich, and an epidermal growth factor (EGF)-like domains, a transmembrane region, and a cytoplasmic tail. ADAMs are widely distributed in many organs, tissues, and cells, such as brain, testis, epididymis, ovary, breast, placenta, liver, heart, lung, bone, and muscle. These proteins are capable of four potential functions: proteolysis, adhesion, fusion, and intracellular signaling. Because the number of ADAM genes has grown rapidly and the biological functions of most members are unclear, this review analyzes the protein structures and functions, their activation and processing, their known and potential activities, and their evolutionary relationships. A sequence alignment of human ADAMs is compiled and their homology and physical data are calculated. The conceivable functions of ADAMs in reproduction, development, and diseases are also discussed.

Cryptosporidium parvum sporozoite pellicle antigen recognized by a neutralizing monoclonal antibody is a beta-mannosylated glycolipid.

The protozoan parasite Cryptosporidium parvum is an important cause of diarrhea in humans, calves, and other mammals worldwide. No approved vaccines or parasite-specific drugs are currently available for the control of cryptosporidiosis. To effectively immunize against C. parvum, identification and characterization of protective antigens are required. We previously identified CPS-500, a conserved, neutralization-sensitive antigen of C. parvum sporozoites and merozoites defined by monoclonal antibody 18.44. In the present study, the biochemical characteristics and subcellular location of CPS-500 were determined. CPS-500 was chloroform extractable and eluted with acetone and methanol in silicic acid chromatography, consistent with being a polar glycolipid. Following chloroform extraction and silicic acid chromatography, CPS-500 was isolated by high-pressure liquid chromatography for glycosyl analysis, which indicated the presence of mannose and inositol. To identify which component of CPS-500 comprised the neutralization-sensitive epitope recognized by 18.44, the ability of the monoclonal antibody to bind CPS-500 treated with proteases, or with alpha- or beta-glycosidases, was determined. Monoclonal antibody 18.44 did not bind antigen treated with beta-D-mannosidase but did bind antigen treated with alpha-D-mannosidase, other alpha- or beta-glycosidases, or a panel of proteases. These data indicated that the target epitope was dependent on terminal beta-D-mannopyranosyl residues. By immunoelectron microscopy, 18.44 binding was localized to the pellicle and an intracytoplasmic tubulovesicular network in sporozoites. Monoclonal antibody 18.44 also bound to antigen deposited and released onto substrate over the course travelled by gliding sporozoites and merozoites. Surface localization, adhesion and release during locomotion, and neutralization sensitivity suggest that CPS-500 may be involved in motility and invasion processes of the infective zoite stages.

Identification, characterization, and relatedness of luteovirus isolates from forage legumes.

ABSTRACT Virus isolates from forage legumes collected from eight different states were identified as luteoviruses closely related to soybean dwarf luteovirus dwarfing (SbDV-D) and yellowing (SbDV-Y) described in Japan. All isolates produced reddened leaf margins in subterranean clover and were transmitted in a persistent manner by Acrythosiphon pisum, but not by Aulacorthum solani. Specific monoclonal antibodies raised against SbDV-Y were differentially reactive with endemic isolates. Immunoblots probed with a SbDV-D polyclonal antiserum showed single 26-kDa coat protein bands, confirming close serological relatedness to SbDV. Analyses of genomic and subgenomic double-stranded RNAs and northern blot analyses confirmed genomic relatedness to SbDV. Based on our results, we conclude that the U.S. luteovirus isolates studied comprise a strain or strains of the soybean dwarf virus that have clovers as common hosts and the pea aphid as a common vector.

Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of human immunodeficiency virus-1 infectivity in vitro.

Heparins/heparan sulfates modulate the function of proteins and cell membranes in numerous biological systems including normal and disease processes in humans. Heparin has been used for many years as an anticoagulant, and anticoagulant heparin-mimetics were developed several decades ago by chemical sulfation of non-mammalian polysaccharides, e.g., an antithrombotic sulfated xylan. This pharmaceutical, which comprises a mixture of sulfated oligoxylans, also mimics most other biological actions of natural heparins in vitro, including inhibition of the human immunodeficiency virus, but the molecular basis for these actions has been unclear. Here, numerous Components of the sulfated oligoxylan mixture were isolated and when bioassayed in the case of anti-HIV-1 infectivity revealed that a structural specificity underlines the capacity of sulfated xylan to inhibit HIV-1, rather than a non-specific mechanism. Components were isolated by chromatographic fractionation through Bio-Gel P10 in 0.5 M ammonium bicarbonate. This fractionation revealed an elution range associated with apparent molecular weights of approximately 22000 to <1500 relative to standard heparin and heparan sulfates and newly prepared sulfated oligosaccharide standards. Components were characterized by metachromatic absorption spectroscopy, ultracentrifugation, GlcA analysis, and potency against HIV-1 infectivity, both in the tetrazolium cytotoxicity assay and in syncytium-forming assays, in CD4-lymphocytes. Structural specificity was indicated by the differential potencies exhibited by the Components: Highest activity (cytotoxicity) was exhibited by Components in the chromatographic region > or = approximately 5500 in mass (50% effective (inhibitory) concentration = 0.5-0.7 microg ml(-1) in the first fractionation series, and 0.1-0.5 microg ml(-1) in a second series). The potency declined sharply below approximately 5400 in mass, but with an exception; a second structure exhibiting relatively high potency eluted among low-mass oligosaccharides which had an average size of approximately a nonomer. Components displayed differential potencies also against the syncytium-forming infectivity of HIV-1. The high potency against syncytium-formation was retained by Components down to a minimum size of about 4500 in mass, smaller than the > or = approximately 5400 required above. One in ten of the beta1,4-linked xyloses in the native xylan are substituted with a monomeric alpha1,2 DGlcA branch. We have speculated that pharmaceutical actions of sulfated xylan might be related to structures involving the alpha-D linked substituents and this was examined using a space-filling model of a sulfated octaxylan and by analyses of Components for GlcA content. Understanding structure/function relations in the heparin-like actions of these agents would be of general significance for the careful examination of their potential clinical usefulness in many human processes modulated by heparins, including AIDS.

Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of Cryptosporidium parvum sporozoites and merozoites.

The apicomplexan protozoan parasite Cryptosporidium parvum causes a diarrheal disease in humans and other mammals for which specific therapy and immunoprophylaxis are unavailable. Passive immunization with Abs against whole C. parvum organisms has variable efficacy in immunocompromised or neonatal hosts. Because apical and surface-exposed zoite Ags of the Apicomplexa are critical to infectivity and targets of protective immunity, we examined the ability of mAbs generated against such Ags in C. parvum sporozoites to passively protect against infection and identify biologically relevant parasite molecules. A panel of mAbs was produced against affinity-purified native Ags using sporozoite apical- and surface-reactive mAb C4A1 as binding ligand. One resulting mAb, designated 3E2, elicited prominent morphologic changes in sporozoites and merozoites characterized by rapid and progressive formation, posterior movement, and release of membranous Ag-mAb precipitates. These changes had a striking resemblance to the malarial circumsporozoite precipitate (CSP) reaction. Sporozoite infectivity was completely neutralized after in vitro exposure to 3E2 and the CSP-like reaction. Furthermore, orally administered 3E2 completely prevented or markedly reduced infection in neonatal BALB/c mice. 3E2 bound to apical complex and surface molecules of zoites and was demonstrated in membranous precipitates by immunoelectron microscopy. In Western blots, 3E2 recognized multiple 46 to approximately 770 kDa sporozoite Ags and an approximately 1300-kDa Ag designated CSL, also expressed by merozoites. CSL was characterized as a soluble glycoprotein exoantigen released by infectious sporozoites. Further, CSL was determined to be the molecular species mechanistically involved in the CSP-like reaction by its identification in SDS-PAGE gels and Western blots of purified membranous precipitates. These findings indicate that CSL has a functional role in sporozoite infectivity and is a candidate molecular target for passive or active immunization against cryptosporidiosis.

Biophysical characterization of a recombinant soluble interleukin 2 receptor (Tac). Evidence for a monomeric structure.

The interleukin 2 receptor (IL2R) plays a prominent role in the biology of T cells, B cells, and NK cells during activation. Of the three chains described, the alpha-chain of the receptor (Tac; IL2R alpha; CD25) is the most subject to regulation and is shed from the surface of activated cells to generate a soluble form in serum and tissues. Conflicting results have been reported on the native structure of soluble Tac, suggesting variously a monomer, a dimer, or higher noncovalent forms, spawning different models for its mechanism of action. We similarly show a large M(r)(app) by HPLC sieving chromatography, suggesting a tetrameric form. However, stoichiometry-ordered size (SOS) analysis of antibody-antigen complexes indicated only a single epitope per Tac molecule, compatible with a monomeric form. This larger M(r)(app) also conflicted with prior in vivo data showing rapid filtration of soluble Tac through the renal glomerulus that was not expected of a larger complex. Using different solvents, denaturants, and columns in the chromatography suggested that the elevated M(r)(app) values were an artifact of solute-column interactions, termed "ionic exclusion", rather than reflecting larger native structures. Analytical ultracentrifugation using a new type of analysis specific to glycoproteins demonstrated monomeric masses under all salt conditions with no tendency to form dimers or higher aggregates. Finally, circular dichroism spectroscopy showed no salt-dependent changes to suggest conformational alterations that might correlate with mobility changes on high pressure liquid chromatography. We conclude therefore that Tac is monomeric under physiologic conditions. Assessments of higher molecular weight for the purified soluble protein by other methods may be explained by the highly acidic nature of the molecule, which hampers matrix penetration with chromatographic media and by the high carbohydrate content and low partial specific volumes that accelerate the molecule in sedimentation media relative to pure protein standards.

Three isoforms of human myelin basic protein: purification and structure.

Myelin basic protein (MBP) occurs in multiple forms. Three of these isoforms from human MBP (HMBP) have been highly purified. HMBP, component 1 (18.5 kDa HMBP-1), was purified by ion-exchange chromatography at pH 10.6 in 2 M urea. During this ion-exchange chromatography, a fraction (Fraction 3), which contained HMBP component 3 (monophosphorylated or deamidated 18.5 kDa) and 17.2 kDa HMBP, was collected and further purified by fast protein liquid chromatography, which separated 17.2 kDa HMBP and HMBP component 3. When the latter was subjected to limited thrombic digestion, all of HMBP component 3 not phosphorylated at theonine 98 was cleaved. This digestion mixture was separated on Sephadex, and yielded pure component 3, monophosphorylated at theonine 98 (HMBP 3pT98), for which phosphate analysis yielded approximately 1 mole P/mole protein, and NMR showed only one phosphorylation site present. Circular dichroism (CD) studies were carried out on dilute solutions of HMBP-1 (18.5 kDa), 17.2 kDa HMBP, and HMBP3pT98 (phosphorylated 18.5 kDa). The CD spectrum of HMBP-1 was similar to that reported for rabbit MBP-1 and bovine MBP-1, but the spectra of 17.2 kDa HMBP and HMBP 3pT98 were distinctly different from HMBP-1. When analyzed by best-fit computations, 17.2 kDa HMBP showed about a 9% increase of ordered structure, and a greater increase, about 12%, was estimated for HMBP3pT98, attributable to beta-structure and beta turn.

Relation between structure and immunologic properties of the Vi capsular polysaccharide.

The Vi capsular polysaccharide of Salmonella typhi is a linear homopolymer of poly-alpha(1----4)GalNAcp variably O acetylated at the C-3 position. Serum antibodies elicited by this antigen confer protective immunity against typhoid fever. The relation between the immunologic properties and structure of Vi was investigated by carboxyl reduction, O deacetylation, and acid hydrolysis. The immunogenicity of Vi was closely related to its degree of O acetylation. Partial O deacetylation slightly increased immunogenicity; complete O deacetylation eliminated the immunogenicity of Vi. O-deacetylated Vi, however, still reacted with antisera prepared by injection of whole bacteria. Carboxyl reduction, in contrast, had a comparatively slight effect upon both the immunogenicity and antigenicity of Vi. Retention levels of antigenicity after acid treatment were greater for both the native and carboxyl-reduced Vi than for the O-deacetylated product. The Courtauld-Koltun space-filling model of a pentamer of Vi demonstrated that the bulky nonpolar O-acetyls, which protrude in rows on both sides, make up most of the surface. The carboxyls are less exposed and are partially shielded by the O-acetyls. The molecular model thus provides an explantation for the dominant role of the O-acetyls, as well as the lesser effect of carboxyl reduction, upon the immunologic properties of Vi.

Role of phosphorylation in conformational adaptability of bovine myelin basic protein.

Controlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated form(s) of component 1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phosphorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in beta-structure(s).

Application of optical properties of the Vi capsular polysaccharide for quantitation of the Vi antigen in vaccines for typhoid fever.

The capsular polysaccharide of Salmonella typhi and of Citrobacter freundii (Vi) is a linear homopolymer of alpha 1,4-linked N-acetylgalactosaminuronic acid, variably O-acetylated at the C-3 position. Vaccines composed of Vi confer protection against typhoid fever with an efficacy of about 70%; Vi has recently been conjugated to proteins to increase its immunogenicity and effectiveness (I.L. Acharya, R. Tapa, V.L. Gurubacharya, M.B. Shrestha, C.U. Lowe, D.D. Bryla, R. Schneerson, J.B. Robbins, T. Crampton, B. Trollfors, M. Cadoz, D. Schulz, and J. Armand, N. Engl. J. Med. 317:1101-1104, 1987; K.P. Klugman, I. Gilbertson, H.J. Kornhof, J.B. Robbins, R. Schneerson, D. Schulz, M. Cadoz, and J. Armand, Lancet ii:1165-1169, 1987; S.C. Szu, A.L. Stone, J.D. Robbins, R. Schneerson, and J.B. Robbins, J. Exp. Med. 166:1510-1524, 1987). Vi, however, cannot be measured by conventional colorimetric methods. Two optical techniques were adapted to quantitate Vi in vaccines. The first, Fourier-transformed infrared spectroscopy, was performed on salt-free, freeze-dried samples. The intensities of the absorbance peaks of Vi were proportional to the amount of Vi within the range of 0.25 to 2.0 mg. The amount of Vi was determined from integrated absorptions at the 1,235- or 1,417-cm-1 band. The second technique, spectrophotometric titration, was more sensitive than the Fourier-transformed infrared spectroscopy and could be performed on dilute solutions. The metachromatic effect of the reaction between the aromatic cationic dye acridine orange and the carboxyl groups of Vi was quantitative within +/- 2% in the range of 20 to 700 micrograms of Vi per ml. The accuracy of the titration of Vi in the vaccines was within +/- 8%. These two methods may be applicable to measure other capsular polysaccharides in vaccines.