Neurochemical and neuroinflammatory perturbations in two Gulf War Illness models: Modulation by the immunotherapeutic LNFPIII.

Gulf War Illness (GWI) manifests a multitude of symptoms, including neurological and immunological, and approximately a third of the 1990-1991 Gulf War (GW) veterans suffer from it. This study sought to characterize the acute neurochemical (monoamine) and neuroinflammatory profiles of two established GWI animal models and examine the potential modulatory effects of the novel immunotherapeutic Lacto-N-fucopentaose III (LNFPIII). In Model 1, male C57BL/6 J mice were treated for 10 days with pyridostigmine bromide (PB) and permethrin (PM). In Model 2, a separate cohort of mice were treated for 14 days with PB and N,N-Diethyl-methylbenzamide (DEET), plus corticosterone (CORT) via drinking water on days 8-14 and diisopropylfluorophosphate (DFP) on day 15. LNFPIII was administered concurrently with GWI chemicals treatments. Brain and spleen monoamines and hippocampal inflammatory marker expression were examined by, respectively, HPLC-ECD and qPCR, 6 h post treatment cessation. Serotonergic (5-HT) and dopaminergic (DA) dyshomeostasis caused by GWI chemicals was apparent in multiple brain regions, primarily in the nucleus accumbens (5-HT) and hippocampus (5-HT, DA) for both models. Splenic levels of 5-HT (both models) and norepinephrine (Model 2) were also disrupted by GWI chemicals. LNFPIII treatment prevented many of the GWI chemicals induced monoamine alterations. Hippocampal inflammatory cytokines were increased in both models, but the magnitude and spread of inflammation was greater in Model 2; LNFPIII was anti-inflammatory, more so in the apparently milder Model 1. Overall, in both models, GWI chemicals led to monoamine disbalance and neuroinflammation. LNFPIII co-treatment prevented many of these disruptions in both models, which is indicative of its promise as a potential GWI therapeutic.

Expert elicitation for deriving input data for probabilistic risk assessment of shipwrecks.

The necessity of having a process in place for adequate risk assessment of shipwrecks that pose a threat to the marine environment is today internationally acknowledged. However, retrieving the desired data for such a risk assessment can prove challenging. One means of addressing this problem is to make use of experts' knowledge and experience. The purpose of this paper is therefore to present and analyse data for risk assessment of shipwrecks derived by expert elicitation. The main outcome is the experts' estimations of (i) the generic probability of an opening in a shipwreck due to the occurrence of a number of activities and (ii) estimations of the degree to which site-specific and wreck-specific indicators affect the probability of opening. Results show that the derived information is applicable in probabilistic shipwreck risk assessment and that the VRAKA framework now contains needed information for integrating generic and site-specific information using Bayesian updating.

Bayesian updating in a fault tree model for shipwreck risk assessment.

Shipwrecks containing oil and other hazardous substances have been deteriorating on the seabeds of the world for many years and are threatening to pollute the marine environment. The status of the wrecks and the potential volume of harmful substances present in the wrecks are affected by a multitude of uncertainties. Each shipwreck poses a unique threat, the nature of which is determined by the structural status of the wreck and possible damage resulting from hazardous activities that could potentially cause a discharge. Decision support is required to ensure the efficiency of the prioritisation process and the allocation of resources required to carry out risk mitigation measures. Whilst risk assessments can provide the requisite decision support, comprehensive methods that take into account key uncertainties related to shipwrecks are limited. The aim of this paper was to develop a method for estimating the probability of discharge of hazardous substances from shipwrecks. The method is based on Bayesian updating of generic information on the hazards posed by different activities in the surroundings of the wreck, with information on site-specific and wreck-specific conditions in a fault tree model. Bayesian updating is performed using Monte Carlo simulations for estimating the probability of a discharge of hazardous substances and formal handling of intrinsic uncertainties. An example application involving two wrecks located off the Swedish coast is presented. Results show the estimated probability of opening, discharge and volume of the discharge for the two wrecks and illustrate the capability of the model to provide decision support. Together with consequence estimations of a discharge of hazardous substances, the suggested model enables comprehensive and probabilistic risk assessments of shipwrecks to be made.

A fault tree model to assess probability of contaminant discharge from shipwrecks.

Shipwrecks on the sea floor around the world may contain hazardous substances that can cause harm to the marine environment. Today there are no comprehensive methods for environmental risk assessment of shipwrecks, and thus there is poor support for decision-making on prioritization of mitigation measures. The purpose of this study was to develop a tool for quantitative risk estimation of potentially polluting shipwrecks, and in particular an estimation of the annual probability of hazardous substance discharge. The assessment of the probability of discharge is performed using fault tree analysis, facilitating quantification of the probability with respect to a set of identified hazardous events. This approach enables a structured assessment providing transparent uncertainty and sensitivity analyses. The model facilitates quantification of risk, quantification of the uncertainties in the risk calculation and identification of parameters to be investigated further in order to obtain a more reliable risk calculation.

(1)H NMR studies of hydroxy protons of the V[beta-Gal(1-->3)-alpha-GalNAc(1-->O)]THPGY glycopeptide.

The hydroxy protons of the disaccharide moiety in the glycopeptide Val-[beta-Gal(1-->3)-alpha-GalNAc(1-->O)]-Thr-His-Pro-Gly-Tyr (1) have been investigated in aqueous solution using (1)H NMR spectroscopy. The chemical shifts (delta), coupling constants ((3)J(CH,OH)), temperature coefficients (d delta/dT), exchange rates (k(ex)), and NOEs have been measured. The data show that the O(2')H of Gal has a reduced contact with water due to steric interference caused by the 2-acetamido group of GalNAc. No interaction, in terms of hydrogen bonding exists between the disaccharide and the peptide moieties, but the rotation around the sugar-peptide linkage is restricted.

Increased p53 mutation frequency during tumor progression--results from a breast cancer cohort.

The mutational patterns of the p53 gene for exons 4-9 were analyzed in 30 recurring tumors compared with the p53 status of the corresponding 30 primary breast cancers. The prevalence of p53 mutations was higher, although not statistically significant (P = 0.07), in the evaluable recurring tumors compared with the corresponding primaries, 12 of 29 (41%) versus 7 of 30 (23%). Twenty-one of the patients had unchanged p53 mutation status in the recurring compared with the primary tumors, whereas 8 had an altered mutational status or pattern in the sequential tumor. These findings indicate that p53 mutations may be an important factor for tumor progression in human breast cancer.

Enzymatic mutation detection method evaluated for detection of p53 mutations in cDNA from breast cancers.

BACKGROUND: Rapid, reproducible, and easily run methods with high sensitivity and specificity are required for mutation screening of clinical samples. We evaluated the Enzymatic Mutation Detection (EMD(TM)) method by analysis of archival cDNA from 203 breast cancer patients and comparison with results of cDNA-based sequencing of the tumor suppressor gene p53. METHODS: The EMD technology uses the T4 endonuclease VII, which cleaves double-stranded DNA at sites where a DNA mismatch is present because of mispairing or an insertion/deletion of nucleotides. The EMD analyses were carried out by dividing the p53 gene into two overlapping fragments that were analyzed separately. After PCR amplification, the fragments were hybridized with wild-type p53 and subsequently exposed to the EMD enzyme. Cleavage products were analyzed and scored using an ALF(TM) automated DNA sequencer and ALFwin Fragment Analyzer software (VER: 1.02). RESULTS: The EMD technique had sensitivities of 45% and 64% and specificities of 83% and 84% for the two fragments, respectively. Patients with EMD-positive, wild-type p53 tumors had a survival similar to that of patients with EMD-negative, wild-type p53 tumors. Node-positive patients with p53 mutated tumors according to sequencing had a statistically significantly worse overall survival than those with p53 wild-type tumors (P = 0.016), whereas this difference in survival was not detected when p53 status was determined with EMD (P = 0.47). CONCLUSIONS: EMD had insufficient sensitivity for consideration in screening for the p53 gene in this archival material. Sequencing must still be considered as the standard procedure.

Hydroxy protons in conformational study of a Lewis b tetrasaccharide derivative in aqueous solution by NMR spectroscopy.

The 1H NMR chemical shifts, vicinal coupling constants, temperature coefficients, and exchange rates of the hydroxy protons of a Lewis b tetrasaccharide derivative, alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> 3)[alpha-L-Fucp-(1 --> 4)]-beta-D-GlcpNAc-1-O(CH2)2NHCOCHCH2, have been measured in aqueous solution. The data did not show any evidence for persistent hydrogen bonds participating in the stabilization of the structure. While most of the hydroxy proton signals have chemical shifts similar to those of the corresponding methyl glycosides, four of them, O(3)H, O(4)H, and O(6)H of Galp, and O(2)H of the Fucp linked to GlcpNAc, exhibit large upfield shifts. This shielding effect has been attributed to the orientation of the hydroxy protons toward the amphiphilic region constituted by the hydroxy groups of the Galp residue and mainly the ring and methyl hydrogens of the Fucp unit attached to the GlcpNAc. The close face to face stacking interaction between the Fucp linked to the GlcpNAc and the Galp residues, as well as the steric interaction between the Fucp linked to the Galp and the GlcpNAc are confirmed by the additional inter-residue NOEs of the exchangeable protons in sugar units which are not directly connected.

New derivatives of reducing oligosaccharides and their use in enzymatic reactions: efficient synthesis of sialyl Lewis a and sialyl dimeric Lewis x glycoconjugates.

The reducing oligosaccharides lactose and lacto-N-tetraose were reductively aminated with benzyloxycarbonylaminoaniline and sodium cyanoborohydride, followed by treatment of the resulting secondary amines with acetic anhydride. The resulting N-acetyl-N-(4-benzyloxycarbonylaminophenyl)-1-amino-1-deoxyaldi tol oligosaccharide derivatives were subjected to stepwise enzymatic elongation with various glycosyltransferases/nucleotide sugars. Purification of the products after each enzymatic step was conveniently performed by solid-phase extraction on silica gel C-18 cartridges. Two oligosaccharide derivatives (with sialyl Lewis a and sialyl dimeric Lewis x structures) were prepared. Conversion of the obtained derivatives into neoglycoproteins by the sequence hydrogenolysis, thiophosgene treatment, and protein coupling was carried out.

Synthesis of a spacer-containing disaccharide fragment of Bordetella pertussis lipopolysaccharide.

The disaccharide 2-(p-aminophenyl)ethyl 4-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-2,3-diacetamido-2 ,3-dideoxy-alpha-D-mannopyranoside uronate, which is assumed to be a partial structure of the Bordetella pertussis polysaccharide, was synthesized starting from D-glucose and D-glucosamine, respectively. The major synthetic transformations were conversion of D-glucosamine into the donor ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-beta-D-glucopyranoside and conversion of glucose, by a sequence involving 2,3-epoxide formation/opening, nucleophilic triflate displacement in the 3-position, and necessary protecting group manipulations, into the acceptor 2-(p-trifluoroacetamidophenyl)ethyl 6-O-benzyl-2,3-diazido-2,3-dideoxy-alpha-D-mannopyranoside. Coupling of the donor and acceptor units promoted by dimethyl(methylthio)sulfonium triflate followed by selective oxidation of the 6'-position and deprotection gave the target disaccharide.

High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.

We have expressed the Neisseria meningitidis lgtA gene at a high level in Escherichia coli. The encoded beta-N-acetylglucosaminyltransferase, referred to as LgtA, which in the bacterium is involved in the synthesis of the lacto-N-neo-tetraose structural element of the bacterial lipooligosaccharide, was obtained in an enzymatically highly active form. This glycosyltransferase appeared to be unusual in that it displays a broad acceptor specificity toward both alpha- and beta-galactosides, whether structurally related to N- or O-protein-, or lipid-linked oligosaccharides. Product analysis by one- and two-dimensional 400 MHz 1H- and 13C-NMR spectroscopy reveals that LgtA catalyzes the introduction of GlcNAc from UDP-GlcNAc in a beta 1-->3-linkage to accepting Gal residues. The enzyme can thus be characterized as a UDP-GlcNAc:Gal alpha/beta-R beta 3-N-acetylglucosaminyltransferase. Although lactose is a highly preferred acceptor substrate the recombinant enzyme also acts efficiently on monomeric and dimeric N-acetyllactosamine revealing its potential value in the synthesis of polylactosaminoglycan structures in enzyme assisted procedures. Furthermore, LgtA shows a high donor promiscuity toward UDP-GalNAc, but not toward other UDP-sugars, and can catalyze the introduction of GalNAc in beta 1-->3-linkage to alpha- or beta-Gal in the acceptor structures at moderate rates. LgtA therefore shows promise to be a useful catalyst in the preparative synthesis of both GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.

Enzymatic glycosylation of reducing oligosaccharides linked to a solid phase or a lipid via a cleavable squarate linker.

Reducing oligosaccharides were converted into their corresponding glycosylamines, and these were reacted with 3,4-diethoxy-3-cyclobuten-1,2-dione (squaric acid diethyl ester). The resulting derivatives could be linked to amino-functionalized lipids, solids, or proteins. Treatment of the obtained lipid or solid conjugates with aqueous bromine or, alternatively, with ammonia-ammonium borate cleaved the linkage and regenerated the oligosaccharide glycosylamines, which were in turn rapidly hydrolyzed to the reducing oligosaccharides. To demonstrate the usefulness of this linkage in enzymatic oligosaccharide synthesis, lactose was linked to a lipid or a solid phase, the obtained conjugates were then subjected to two enzymatic glycosylations (either consecutively or 'one-pot'). The resulting materials were then cleaved to give, in both cases, the expected reducing tetrasaccharide (lacto-N-neotetraose) in good yield.

Contulakin-G, an O-glycosylated invertebrate neurotensin.

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(beta-->3) GalNAc(alpha-->)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10 contulakin-G was approximately an order of magnitude lower than that of neurotensin1-13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.

Reference values of facial features in Scandinavian children measured with a range-camera technique.

Reference values of facial features in white Scandinavian children were ascertained using a newly developed range-camera technique with a three-dimensional measuring program specifically designed for anthropometric measurements. Seven facial features in 613 healthy subjects (322 girls, 291 boys), aged 1 month to 18 years, and nine subjects with the fetal alcohol syndrome,aged 7 to 18 years, were studied. Data were analysed by multivariate multiple regression and measurements of each facial feature plotted against age and presented as curves, with 95% and 99% univariate prediction limits. All children with fetal alcohol syndrome had shorter palpebral fissures, and in six of them the outer canthal distance was shorter than that in the reference group. The reference values presented may be useful in clinical practice--for example, in the evaluation of children with syndromes that result in dysmorphology of the face and in the planning of plastic and reconstructive surgery.

Comparison between p53 protein measurements using the luminometric immunoassay and immunohistochemistry with detection of p53 gene mutations using cDNA sequencing in human breast tumors.

The p53 mutational status of 226 representative primary breast cancer samples, derived from a population-based cohort, was analyzed using cDNA-based sequencing. The results were compared with those obtained with immunohistochemistry (IHC) on microwave-treated paraffin sections and the p53 specific luminometric immunoassay (LIA) on cytosols, all from the same individuals. Thirty-seven mutations were found using cDNA sequencing and were categorized into A) missense mutations in the evolutionarily conserved regions; B) missense mutations outside the evolutionarily regions; and C) deletions, insertions and nonsense mutations. Using optimal cut-off values, LIA detected 15 of 16 missense mutations in category A, in which IHC detected all 16. In category B, 10 of 13 and 7 of 13 mutations were detected, respectively. Some of the samples in category A had a very high p53 protein content when measured with the LIA, the reason for this being discussed. IHC detected 0 of 5 stop codon and 0 of 3 deletions/insertions mutations, while the LIA method detected 2 of 5 stop codon mutations and 1 of 3 deletion/insertion mutations. Compared with cDNA sequencing, protein analyses using optimal cut-off values resulted in an overall sensitivity and specificity of 64.9% and 89.9%, respectively, for the LIA method. Corresponding values were 72.2% and 92% for IHC. In addition, patients from whom p53 mutations could be detected by cDNA sequencing had a statistically significant (p = 0.0137) shorter survival, which was not readily apparent using the alternative LIA or IHC approaches at optimal cut-off values.

Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets.

The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.

Solid-Phase Enzymatic Synthesis of a Sialyl Lewis X Tetrasaccharide on a Sepharose Matrix.

Thiopyridyl Sepharoses with different linker arm lengths were prepared from epoxy Sepharose 6B by reaction first with 1,8-diamino-3,6-dioxaoctane and then with, sucessively, diethoxy-3-cyclobutene-1,2-dione (squaric acid diethyl ester) and 1,8-diamino-3,6-dioxaoctane in several cycles, followed by reaction of the obtained amino Sepharoses with, successively, thiobutyrolactone and 2,2'-dithiopyridine. The thiopyridyl Sepharoses were reacted with the glucosamine derivative 2-(3'-mercaptobutyrylamido)ethyl 2-acetamido-2-deoxy-beta-D-glucopyranoside, giving GlcNAc Sepharoses with different linker lengths. Enzymatic galactosylation of these with beta-(1-4)-galactosyltransferase and UDP-galactose gave yields varying between 70 and 98%, and there was a clear correlation between linker length and yield. A GlcNAc Sepharose with a long linker was then used in a solid-phase synthesis of a sialyl Le x tetrasaccharide. The three required enzymes (galactosyl-, sialyl, and fucosyltransferase) and nucleotide sugars were reacted consecutively with the GlcNAc Sepharose, giving, after cleavage from Sepharose with DTT, the free sialyl Le x tetrasaccharide derivative in a 57% total yield after purification.

Genes involved in cell cycle G1 checkpoint control are frequently mutated in human melanoma metastases.

A common characteristic of cancer cells is unrestrained cell division. This may be caused by mutational changes in genes coding for components of cell cycle-controlling networks. Alterations in genes involved in G1 checkpoint control have been registered in many human tumours, and investigations from several laboratories show that such alterations, taken together, are the most frequent changes detected in cancer cells. The present paper describes mutational analysis by polymerase chain reaction-single-strand conformation polymorphism (PCR/SSCP) and nucleotide sequence analysis of the genes coding for the p15, p53 and N-ras proteins in 26 metastases from 25 melanoma patients. The registered mutation frequencies add together with previously registered mutations in p16 in the same patient samples to a substantial total frequency of 44% of patients with mutation in at least one of the investigated genes. These results show the occurrence of heterogeneous defects among components of the cell cycle controlling machinery in a human melanoma tumour sample collection and demonstrate that the total frequency of detected alterations increases with the number of cell cycle controlling genes included in the screening panel.

The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry.

BACKGROUND: Mutations in the p53 tumor suppressor gene (also known as TP53) have been detected in a wide variety of human cancers. In breast cancer, the presence of p53 gene alterations has been associated with worse prognosis. PURPOSE: We compared a complementary DNA (cDNA)-based sequencing method and an immunohistochemical (IHC) method for their abilities to detect p53 mutations in breast cancer specimens. In addition, we determined the prognostic value of information obtained when these two methods were used. METHODS: Specimens from 316 primary breast tumors were evaluated for the presence of mutant p53 protein by use of the mouse monoclonal antibody Pab 1801 (that recognizes both wild-type and mutant forms of p53) and standard IHC methods. In addition, the entire coding region of p53 genes expressed in these tumors was screened for mutations by combining reverse transcription, the polymerase chain reaction, and DNA sequencing. Probabilities for overall survival (OS), breast cancer-corrected survival (BCCS; death from breast cancer is the considered event), and relapse-free survival (RFS) were estimated by use of the Kaplan-Meier method, and survival curves for different patient subgroups were compared by use of the logrank method. All reported P values are from two-sided tests. RESULTS: Sixty-nine (22%) of 316 tumors had p53 gene mutations detected by the cDNA-based sequencing method; only 31 (45%) of these mutations were located in evolutionarily conserved portions of the p53 coding region. Sixty-four tumors (20% of the total) had elevated levels of p53 protein as detected by IHC, suggesting the presence of mutations. Of the sequencing-positive tumors (i.e., p53 mutant), 23 exhibited negative IHC reactions, indicating that IHC failed to detect 33% of the mutations. Furthermore, 19 of the IHC-positive tumors were sequencing negative (i.e., p53 wild-type), suggesting a 30% false-positive frequency with IHC. Four tumors (1.3% of the total) could not be analyzed by the cDNA-based sequencing method, and three tumors (1% of the total) could not be analyzed by IHC. The 5-year estimates for RFS, BCCS, and OS were significantly shorter for patients with p53 sequencing-positive tumors than for patients with sequencing-negative tumors (P = .001, P = .01, and P = .0003, respectively). Patients with IHC-positive tumors showed reduced survival in all three categories when compared with those with IHC-negative tumors, but the differences were not statistically significant. CONCLUSIONS: Use of a cDNA-based sequencing method to determine the status of the p53 gene in primary breast cancers yielded better prognostic information than IHC performed with the Pab 1801 monoclonal antibody.

Overview on human breast cancer with focus on prognostic and predictive factors with special attention on the tumour suppressor gene p53.

A long list of potential prognostic markers has been analysed for breast cancer, some of them will be reviewed in this article. The lymph node status is still the best prognostic marker. The lymph node status combined with information on tumour size, receptor- and proliferation status of the tumour should be analysed as standard for all breast cancer patients. Prognostic information for breast cancer patients has also been described for the membrane protein c-erbB2, the protease cathepsin D, plasminogen activators and inhibitors, certain oncogenes and tumour suppressor genes. Some of these factors also give potential additional information on the response to different oncological therapies, and are better denoted predictive factors. In this overview we shortly describe the above mentioned prognostic factors with major focus on the tumour suppressor gene p53 and its prognostic value and potential predictive value.