Complete sequencing of the p53 gene provides prognostic information in breast cancer patients, particularly in relation to adjuvant systemic therapy and radiotherapy.

The complete coding region of the p53 gene was sequenced from 316 consecutively presented breast cancers, of which 97 were lymph node positive and 206 were node negative. The p53 status was related to prognosis and effect of adjuvant therapy. In all, 69 individual mutations, 29 in node-positive tumours, were demonstrated throughout the whole coding sequence. The mutation sites were partly different for node-positive and node-negative patients. p53 mutations in the evolutionary conserved regions II and V were associated with significantly worse prognosis. Adjuvant systemic therapy, especially with tamoxifen, along with radiotherapy seemed to be of less value to p53 mutation- and lymph node-positive tumours.

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.

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.

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.

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.

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.

p53 Status predicts survival in breast cancer patients treated with or without postoperative radiotherapy: a novel hypothesis based on clinical findings.

PURPOSE AND METHODS: Primary breast cancer tumors without axillary metastases from 206 consecutive patients in a population-based cohort were investigated with regard to the presence of an intact p53 gene using a cDNA-based sequencing method. Clinical follow-up data and outcome of node-negative patients without any adjuvant systemic therapy (n = 168) were related to locoregional radiotherapy and p53 status. RESULTS: Mutations in p53 occurred in 31 node-negative breast cancer patients who did not receive any systemic adjuvant treatment, but were treated with postoperative locoregional radiotherapy or nothing. Node-negative breast cancer patients with p53 mutations had significantly improved relapse-free survival (P = .0007), breast cancer-corrected survival (P = .01), and overall survival (P = .02) rates when treated with locoregional radiotherapy. In node-negative breast cancer patients with wild-type p53, there was no statistically significant difference in outcome between patients who received locoregional radiotherapy and those who did not. Cox proportional hazards models indicate that mutant p53 is associated with worse prognosis independent of response to radiotherapy and that response to radiotherapy is qualitatively different in tumors with p53 mutations compared with those with wild-type p53. CONCLUSION: Our clinical findings define a group of breast cancer patients in whom locoregional radiotherapy improves relapse-free, breast cancer-corrected, and overall survival. The outcome for irradiated node-negative breast cancer patients with p53 alterations indicates that irradiation can induce cell death even in the presence of p53 mutations.

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.

MCH-induced pigment aggregation in teleost melanophores is associated with a cAMP reduction.

It has previously been shown that alpha 2-adrenoceptors are involved in noradrenaline-induced pigment aggregation within fish melanophores. In the present investigation, melanin concentrating hormone (MCH) elicited pigment aggregation (EC50 approximately 1 x 10(-7) M) that was associated with a significant reduction in the cAMP content; 1 x 10(-7) M MCH reduced the cAMP content from a basal level of 50.4 +/- 2.8 pmol/mg protein to 36.9 +/- 3.8 pmol/mg protein. Like the alpha 2-adrenoceptor-induced pigment aggregation, the MCH response was effectively blocked by the adenylate cyclase stimulator forskolin. These findings suggest that attenuation of cAMP may serve as an intracellular signal transduction mechanism for both MCH and noradrenaline.

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.

Synthesis of a dimeric Lewis X hexasaccharide derivative corresponding to a tumor-associated glycolipid.

The dimeric Lewis X hexasaccharide p-trifluoroacetamidophenylethyl O-beta-D-galactopyranosyl-(1----4)-O-[alpha-L-fucopyranosyl-(1----3)]-O- (2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----3)-O-beta-D-galactopyrano syl- (1----4)-O-[alpha-L-fucopyranosyl-(1----3)]-2-acetamido-2-deoxy-beta-D- glucopyranoside (14), which is a derivative of a tumor-associated glycolipid, was synthesized from thioglycoside intermediates. A protected disaccharide was used as a key-intermediate for synthesis of the p-nitrophenylethyl glycoside of suitably protected O-beta-D-Galp-(1----4)-O-beta-D-GlcpN-(1----3)-O-beta-D-Galp-(1--- -4)-beta-D- GlcpN, which, after selective deblocking, was di-L-fucosylated and deprotected to give 14.

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.

Synthesis of the methyl and 1-octyl glycosides of the P-antigen tetrasaccharide (globotetraose).

The methyl and 1-octyl beta-glycosides of the P-antigen tetrasaccharide [globotetraose, beta-D-GalpNAc-(1----3)-alpha-D-Galp-(1----4)-beta-D-Galp-(1----4) -D-Glc] were synthesised from a tetrasaccharide precursor, prepared using methyl disaccharide 1-thioglycosides as intermediates. In the key glycosidation with silver triflate, HO-2 was used as an alpha-directing group in the glycosyl bromide.

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.

(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.

Synthesis of type 2 human blood-group antigenic determinants. The H, X, and Y haptens and variations of the H type 2 determinant as probes for the combining site of the lectin I of Ulex europaeus.

Chemical syntheses of the human blood-group antigenic determinants derived from N-acetyllactosamine are described; namely, the 6-deoxy derivative, the 4'-epimer, and the 5 H type 2 [alpha LFuc(1 to 2)beta DGal-(1 to 4)beta DGlcNAc], X [beta DGal(1 to 4)[alpha LFuc(1 to 3)[beta DGlcNAc], and Y [alpha LFuc-(1 to 2)beta DGal(1 to 4)[alpha LFuc(1 to 3)]beta DGlcNAc] determinants as glycosides of 8-carboxymethyloctanol. In order to study the binding of the H type 2 determinant with the lectin I of Ulex europaeus, structures designed to specifically alter the hydrophilic and hydrophobic portions of the H type 2 determinant were also prepared; namely, the 6-deoxy derivative, the 4'-epimer, and the 5"-nor-homolog. The use of these structures, together with the H type 1 hapten and the N-deacetylated forms of both the H type 1 and H type 2 determinants, as inhibitors of the agglutination of O red cells by the lectin allowed the conclusion that the binding of the H type 2 determinant is hydrophobic; the binding involves a wedge-like portion of the determinant that is basically hydrophobic, except for the 5-hydroxymethyl group, which is at the tip of the wedge and forms an intramolecular hydrogen-bond with O-5 for acceptance by a hydrophobic cleft at the surface of the lectin. Blocking procedures involving alkoxymethyl groups and new experiences involving glycosylation reactions are described.

The conformation of Salmonella O-antigenic polysaccharide chains of serogroups A, B, and D1 predicted by semi-empirical, Hard-Sphere (HSEA) calculations.

The preferred conformation of the tetrasaccharide repeating units of Salmonella Serogroups A, B, and D1 have been calculated. The semiempirical calculations used the Hard-Sphere, Exo-Anomeric (HSEA) approach to derive a conformational model, which could be used to assist the interpretation of conformations significantly populated in aqueous solution. The calculated model was extended to include a pentasaccharide repeating unit bearing an alpha-D-glucopyranose-branch point. The 3,6-dideoxyhexose to D-mannose linkage was shown to possess a steep energy surface with a minimum, which results in good exposure of the dideoxyhexose O-2 and O-4 atoms. Stereochemical changes involving the equatorial or axial disposition of these atoms are the distinguishing structural features of the A, B, and D1 serogroups. A lipophilic surface involving the 6-deoxy groups of the dideoxy-D-hexose and L-rhamnose residues was identified, and the possible implications of these features in antigenic determinants is discussed. The preferred conformation predicted by the HSEA method correlates with the known antigenic specificities of polysaccharides belonging to these Salmonella serogroups.

The conformation of Salmonella O-antigenic oligosaccharides of serogroups A, B, and D1 inferred from 1H- and 13C-nuclear magnetic resonance spectroscopy.

The conformational model derived by the HSEA calculation method was used to interpret the n.m.r. data for solutions of oligosaccharides corresponding to the Salmonella serogroups A, B, and D1 antigenic determinants. The favored conformer, derived by calculation, accounted for the observed, chemical-shift changes and accurately predicted the existence and magnitude of inter-ring proton n.O.e.'s. Extensive proton-density and compression of proton, Van der Waals radii were correlated with deshielding of specific proton-resonances. The model of lipopolysaccharide conformation accounts for the known antigenic properties of Salmonella O-antigens.

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.