A validation study of the clinical diagnosis of Dup15q syndrome: Which symptoms matter most?

PURPOSE: Dup15q syndrome is a rare genetic disease with a fairly nonspecific phenotype, clinical heterogeneity, and a wide spectrum of severity. However, no formal characterization has been attempted to select clusters of symptoms, signs and instrumental tests, to be used in the differential diagnosis with other neurodevelopmental disorders. Thus, our purpose was to identify symptoms, signs and instrumental findings, singly or in various combinations, favoring the early diagnosis of the Dup15q syndrome and the indication for genetic testing. METHODS: 25 patients with Dup15q syndrome and 25 age and sex matched controls with other neurodevelopmental disorders were the study population. Patients' history, clinical and instrumental assessment were examined by five expert child neurologists blind to the genetic diagnosis. Each rater was asked to make the diagnosis in three subsequent steps: 1. Revision of the medical records; 2. Examination of the videorecorded clinical findings; 3. Assessment of the instrumental tests. Inter-rater agreement was measured with the Kendall's coefficient of concordance) and the Kappa statistic. Sensitivity, specificity and predictive values for symptoms, signs and instrumental findings, singly or in various combinations, were measured. RESULTS: The Kendall's coefficient for the diagnosis of Dup15q syndrome was 0.43 at step 1 was 0.43, at step 2 was 0.42, at step 3. Patients with past feeding difficulties, hypotonia during the neonatal period, and epilepsy had >80 % probability of having the Dup15q syndrome. CONCLUSION: Feeding difficulties, hypotonia and epilepsy, though unspecific, can be used as signals of Dup15q syndrome and focused search of genetic abnormalities.

Association among low whole blood viscosity, haematocrit, haemoglobin and diabetic retinopathy in subjects with type 2 diabetes.

OBJECTIVES: Haemorheological variables influence endothelial function through the release of several factors. Clinical studies have described an association among blood viscosity, haematocrit, haemoglobin and macro-angiopathy. Few data are reported about the association between haemorheological variables and micro-angiopathy. The aim of the present study was to evaluate the association between these variables and retinopathy in subjects with type 2 diabetes. METHODS: 111 men, 79 postmenopausal women, and 95 healthy age- and sex-matched controls were recruited. Haematocrit and haemoglobin were measured by standard methods. Blood viscosity was calculated according to the formula (0.12× haematocrit)+(0.17× (plasma proteins-2.07)). Subjects were grouped according to the presence or absence of diabetic retinopathy, while the severity of retinopathy was classified according to the Early Treatment Diabetic Retinopathy Study scale. RESULTS: Haemoglobin, haematocrit and whole blood viscosity were significantly lower in subjects with retinopathy compared to subjects without retinopathy in both sexes. These variables significantly decreased with increasing severity of retinopathy. A multiple logistic regression analysis confirmed the independent inverse association among viscosity, haematocrit, haemoglobin and retinopathy (p<0.01). CONCLUSION: Results demonstrate the association among low viscosity, haemoglobin, haematocrit and diabetic retinopathy. The mechanisms responsible for this association can be hypothesised. Reduced haemoglobin might cause direct organ damage. Low blood viscosity, through the reduction of shear stress, might inhibit the anti-atherogenic functions of endothelial cells.

Interaction of fusarium mycotoxins, fusaproliferin and fumonisin B1, with DNA studied by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) in negative ion mode was used to monitor the possible noncovalent adduct formations between DNA analogue oligonucleotides and two Fusarium mycotoxins, fumonisin B1 and fusaproliferin. Using mild experimental ESI conditions specific noncovalent interactions were detected between both single- and double-stranded model oligonucleotides and fusaproliferin with 1:1 stoichiometry. Similar association complexes were observed for the deacetyl derivative of fusaproliferin. There were no peaks due to adduct formation present in the mass spectra of fumonisin B1, incubated with oligonucleotides in a wide concentration range, suggesting no specific interaction for this molecule. In a competitive complexation reaction, another mycotoxin, the beauvericin, forms more stable association complex with DNA than fusaproliferin. These findings can be of use in the understanding of molecular mechanisms of action during apoptosis and can be correlated with the teratogenic effect of fusaproliferin.

Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines.

A novel method for measuring the antioxidant activity using N, N-dimethyl-p-phenylenediamine (DMPD) was developed. The radical cation of this compound gives a stable colored solution and a linear inhibition of color formation can be observed in the presence of 0. 2-11 microg of TROLOX. The experimental protocol, which is rapid and inexpensive, ensures sensitivity and reproducibility in the measure of antioxidant activity of hydrophilic compounds. The effectiveness of the DMPD method on real foods was verified by evaluating the antioxidant ability of wine samples coming from different areas of Campania, Italy. Antioxidant capacity of wines is strictly related to the amount of phenolic compounds. The results obtained by the DMPD method are very similar to those obtained on the same samples when the radical cation of 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Miller et al., 1996) was used.

LC/MS analysis and antioxidative efficiency of Maillard reaction products from a lactose-lysine model system.

Aqueous solutions of lactose and lysine were refluxed for up to 4 h without pH control. Samples were collected every hour, and the reaction was monitored by measuring the pH, the optical density at 420 nm, and the relative antioxidative efficiency (RAE). The greatest change in optical density and antioxidative efficiency occurred for the mixture heated for 4 h. The 4 h solution was separated into three fractions according to the molecular weights of the components and tested for RAE. The high molecular weight fraction was more colored, and it had the highest antioxidative activity. The low molecular weight fraction was separated by high-performance liquid chromatography (HPLC). RAE values were measured for each purified compound. HPLC coupled with diode array and electrospray mass spectrometry allowed a rapid screening of the solutions and a tentative identification of several peaks. Nuclear magnetic resonance analysis allowed the identification of galactosylisomaltol and pyrraline. The resonance assignments for these compounds were revised.

Polyclonal antibodies against fusaproliferin.

Fusaproliferin (FP), a toxic metabolite of the world-wide maize pathogens Fusarium proliferatum and Fusarium subglutinans, was recently found to be a natural contaminant of maize. Its toxic activity on haematopoietic human cell lines and its teratogenic effects on chicken embryos has been recently proved. Therefore a sensitive, rapid, and inexpensive screening test to detect FP in agricultural commodities is necessary to protect human health. FP-hemiglutarate conjugated to modified bovine serum albumin was synthesized, characterized, and used as an antigen for raising polyclonal antibodies by immunizing rabbits. Indirect and competitive ELISA and immunoblotting analyses were performed to determine antibody specificity towards the mycotoxin. The determination of 10 micrograms of free FP/mL was achieved using antibodies purified by means of affinity chromatography on a FP-lysine-Sepharose column. This unsatisfactory detection limit is due to high background values; thus, this method is not competitive with traditional UV-HPLC methods.

Oxidative stress and antioxidants at skin biosurface: a novel antioxidant from lemon oil capable of inhibiting oxidative damage to the skin.

Atmospheric pollutants are an important source of oxidative and nitrosative stress both to terrestrial plants and to animals. Skin, which has a highly differentiated and certainly complex organizational structure, is particularly vulnerable to free radical damage because of its contact with oxygen and with other environmental stimuli. Fruit and vegetables contain several classes of compounds that when ingested can potentially contribute to antioxidant defenses. In the present study we employed a novel gas chromatographic method to assess the antioxidant properties of a natural compound isolated from lemon oil, which we have called Lem1. We provide experimental evidence that Lem1 is endowed with a strong antioxidant activity and that it is capable of inhibiting free radical-mediated reactions, as evaluated in vitro and in vivo. The present study extends our previous findings and demonstrates that topical application of Lem1 in healthy volunteers significantly increases the antioxidative potential of skin biosurface, thus highlighting the effectiveness of a natural antioxidant biotechnology in the antiaging management of skin.

Identification of a beta-lactoglobulin lactosylation site.

Thermal treatment of milk leads to non-enzymatic glycosylation of proteins through Maillard reaction. Free NH2 groups of basic amino acids react with the reducing carbonyl group of lactose forming the so-called Amadori products. Electrospray mass spectrometry analysis shows that beta-lactoglobulin (beta-LG), the major whey protein, undergoes lactosylation under industrial thermal treatment. In order to investigate the specificity of reactive sites for lactose binding the analysis of trypsin hydrolysates of beta-LG isolated from different industrial milks was performed. Results demonstrate that Lys-100 is a preferential lactosylation site of beta-LG during industrial milk treatment. These results were confirmed by an analysis of the three-dimensional model of the protein which showed that Lys-100 had the highest solvent accessibility and proximity to another amino group making Lys-100 the best candidate to lactosylation. Lys-47, previously identified by other authors, showed a good proximity to another Lys residue, but an intermediate level of exposition to solvent.

Human rod monochromacy: linkage analysis and mapping of a cone photoreceptor expressed candidate gene on chromosome 2q11.

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at theta = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of approximately 3 cM covering the ACHM2 locus for rod monochromacy. Radiation hybrid mapping of the CNGA3 gene encoding the alpha-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen-D2S2222-D2S2175-(D2S2187/D2S2311)-qtel ofmarkers on 2q11 and showed that the CNGA3 gene maps most closely to D2S2187 and D2S2311. These data indicate that the CNGA3 gene maps within the critical interval of the ACHM2 locus for rod monochromacy and thus is a candidate gene for this disease.

Long-term ethanol administration enhances age-dependent modulation of redox state in central and peripheral organs of rat: protection by metadoxine.

Evidence is accumulating that intermediates of oxygen reduction may be associated with the development of alcoholic disease. In addition, free radical-induced perturbation of the oxidant/antioxidant balance in cells is widely recognized as the main causative factor of age-related disorders. In the present work, we investigated the effects of 25 months of ethanol consumption on the antioxidant defense system in different organs of rat in comparison with normal aging, in the absence and presence of treatment with metadoxine, an ion pair composed of pirrolidone carboxylate and pyridoxine. We demonstrate that aged rats underwent a significant perturbation of the antioxidant defense system, as indicated by depletion of reduced glutathione (GSH) content, and increases in oxidized GSH and free radical-induced luminescence associated with a decrease of GSH reductase and an increase of GSH transferase activities. These modifications, observed particularly in the liver and brain with respect to other organs, were enhanced by long-term alcohol exposure, and interestingly, significantly reduced after metadoxine supplementation. Our results indicate that increased GSH transferase activity and decreased GSH reductase activity, followed by thiol depletion, are important factors sustaining a pathogenic role for oxidative stress in aging and in all situations where age-correlated changes occur. Administration of metadoxine greatly reduces these metabolic abnormalities. This evidence supports the pharmacological potential of metadoxine in the management of alcoholic disturbances.

Long-term ethanol administration enhances urinary ultraweak luminescence and age-dependent modulation of redox in central and peripheral organs of the rat.

Numerous experimental evidence sustains a pathogenic role for oxidative stress in aging. Acute and chronic ethanol metabolism is also known to be associated with oxidative perturbation of cellular oxidant/antioxidant balance. In the present work we investigated the effects of 25 months of ethanol consumption on the antioxidant defense system in different organs of rats, in comparison with normal and aged animals. We show that aged rats underwent a significant perturbation of the antioxidant defense system, as indicated by depletion of reduced glutathione content, increases in oxidized glutathione and free radical-induced urinary luminescence associated with a decrease of glutathione reductase and increase of glutathione transferase activities. These modifications, observed particularly in the liver and brain, were enhanced by long-term alcohol exposure. Our results indicate that increased glutathione transferase activity and decreased glutathione reductase activity, followed by thiol depletion, are important factors sustaining a pathogenic role for oxidative stress in aging and in all situations where age-correlated changes occur. They also reinforce the oxidative potential of toxic compounds, such as ethanol intoxication.

Changes in the glycosylation pattern of circulating gonadotropins after acute administration of gonadotropin-releasing hormone in patients with anorexia nervosa.

To study the involvement of gonadotropin-releasing hormone (GnRH) in glycosylation of circulating gonadotropin isoforms in anorexia nervosa (AN), 14 amenorrhoic patients with AN, 14 age-matched volunteers in early follicular phase, and five normal-weight re-fed patients with AN were investigated under baseline conditions and after acute administration of GnRH. Plasma gonadotropins were assayed using IRMA before and after concanavalin A affinity chromatography. Baseline plasma gonadotropin levels were lower for both AN and re-fed AN patients than in controls (P<0.005). The increase in FSH and LH after GnRH administration was lower than in controls for AN (P<0.005) and re-fed AN (P<0.005 and P<0.05 respectively) patients. Percentages of total gonadotropin not bound to concanavalin A (complex carbohydrate chains) under baseline conditions were higher in patients with AN than in controls (P<0.005) but decreased after GnRH administration (P<0.001). In re-fed AN patients, the percentage of unbound FSH was higher than in controls (P<0.05), and decreased after GnRH administration (P<0.001), whereas the percentages of unbound LH were not significantly different from controls either before or after GnRH administration. These data suggest that: (a) the acute administration of GnRH induces quantitative and qualitative changes in circulating gonadotropin isoforms in both normal controls and AN patients; (b) during recovery the LH response in re-fed AN patients is associated with a glycosylation pattern that is the same as that for controls.

Microheterogeneity characterization of a paracelsin mixture from Trichoderma reesei using high-energy collision-induced dissociation tandem mass spectrometry.

The microheterogeneity of the paracelsin mixture broth of Trichoderma reesei was analysed using mass spectrometric methods, in particular high-energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). Based on the liquid secondary ion mass spectrum of the mixture, there are three main components, with molecular masses M and (M +/- 14), together with two minor components of molecular weight (M +/- 28). The high-energy CID tandem mass spectra of both the protonated and sodiated molecules yielded abundant and characteristic fragment ions, but of very different types. It was found that a paracelsin peptaibol in a mixture could be successfully sequenced based on the tandem mass spectra of its protonated and sodiated molecules or, alternatively, on the tandem mass spectra of its y7 and b13 fragment ions. A terminology for indicating these sequential peptide fragments is proposed. To determine the sequence of new analogues, tandem mass spectra of the y7, (y7 +/- 14), b13, (b13 +/- 14) and (MH +/- 14) positive ions were also taken. Based on these experiments, four new paracelsin components (PA-F, PA-G, PA-H and PA-I) were sequenced successfully. The microheterogeneity of the mixture was found to be more pronounced than had been assumed previously. In these new analogues, besides positions 6 and 9, position 17 is also involved in the exchange. MS/MS studies on minor fragment ions, such as (b13-28) and (b8-14) show further microheterogeneity at positions 3, 5, 10 and 12, which increase the number of possible minor components.

Observation of non-covalent interactions between beauvericin and oligonucleotides using electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry has been used to study the possible non-covalent interaction between oligonucleotides and beauvericin (B) mycotoxin. Beauvericin-oligonucleotide adduct formation was observed even at low mycotoxin concentration (25 pmol/microL). Adducts were found with different numbers of B ligands attached. The selectivity of binding of B ligands to two different oligonucleotides has been shown to be similar indicating that beauvericin does not have a strongly preferred base sequence or base site in the DNA. In a competitive complexation reaction, beauvericin forms specific adducts with oligonucleotide while another mycotoxin, nigeromicin, which causes apoptosis without fragmentation of DNA, does not.

Fusaproliferin production by Fusarium subglutinans and its toxicity to Artemia salina, SF-9 insect cells, and IARC/LCL 171 human B lymphocytes.

Fusarium subglutinans is an important pathogen of maize and other commodities worldwide. We examined MRC-115 and 71 other F. subglutinans strains from various geographic areas for their ability to synthesize fusaproliferin, a novel toxic sesterterpene recently isolated from F. proliferatum. Fusaproliferin production ranged from 30 to 1,500 micrograms/g of dried ground substrate, with 33 strains producing more than 500 micrograms/g. In particular, strain MRC-115 produced as much as 1,100 to 1,300 micrograms/g. In toxicity studies of two invertebrate models, fusaproliferin was toxic to Artemia salina (50% lethal dose, 53.4 microM) and to the lepidopteran cell line SF-9 (50% cytotoxic concentration, approximately 70 microM, after a 48-h exposure). Fusaproliferin was also toxic to the human nonneoplastic B-lymphocyte cell line IARC/LCL 171 (50% cytotoxic concentration, approximately 55 microM in culture in stationary phase after a 48-h exposure). Experiments performed will cells exposed at seeding suggested a possible cytostatic effect at subtoxic concentrations.

Structure and absolute stereochemistry of fusaproliferin, a toxic metabolite from Fusarium proliferatum.

Fusaproliferin is a toxic sesterterpene isolated from Fusarium proliferatum, a widespread pathogen of cereals. Its absolute configuration has been determined by single crystal X-ray diffraction analysis. Fusaproliferin is considered to be a sesterterpene with a new ring skeleton having four C = C double bonds and four chiral atoms. The configurations of the four chiral atoms C10, C14, C15, and C19 are (R), (S), (R), and (S), respectively. In the solid state the macrolide shows a concave hydrophobic surface and hydrophilic convex face. The absolute configuration of C14 and C15 is the same as that observed for retigeranic acid, consistent with fusaproliferin being formed via a sesterterpenic-type biosynthetic pathway.

Which kind of surgery in elderly people for colorectal cancer?

Colo-rectal cancers are of high incidence in elderly patients. Different clinical features and the peculiar behavior of the tumor may influence surgical results and should be considered in the decision making, when the surgeon has to decide whether to perform radical gut resection or less straining palliative procedures. In a retrospective study, 102 large bowel cancer patients are analyzed submitted to surgery in the period 1989-1994. Patients were divided in two age classes: Group A: above 70 years of age, 45 cases (44.2%); Group B: under 70 years of age, 57 cases (55.8%). Emergency surgery procedures were necessary in 35 patients (34.4%), 20 cases (57%) in Group A and 15 cases (43%) in Group B. Radical resections could be performed in 25 (37%) old patients, 67% of the cases underwent a curative resection. Perioperative mortality and surgical complication rates were significantly higher in Group A than in Group B. The technical and biological difficulties in performing radical curative resections, the high complication rates and the occurrence of negative results of treatments provide a reason for careful evaluation of the risk/benefit ratio in older patients, where less straining palliative therapies may sometimes offer similar results.

Changes in endogenous lipoperoxidation and antioxidant enzyme status induced by cysteine in the substantia Nigra.

It is generally accepted that reactive oxygen species have a major role in the mediation of cell damage and that free sulfhydryl groups are vital in cellular defence against endogenous or exogenous oxidants. Modification of cellular oxidant/antioxidant balance has been involved in the neuropathogenesis of several diseases, e.g., stroke, Parkinson's disease, Alzheimer's disease and physiological ageing. An increasingly important area of antioxidant defence is based on sulfhydryl chemistry, owing to the role of -SH groups in the function of macromolecular structures such as enzymes and cellular membranes. Thiols, however, may themselves generate deleterious free radicals. In the present study we provide experimental evidence suggesting a selective effect of cysteine in promoting reactions of oxidative stress in the brain areas of substantia nigra and septum, but not in other different areas which were associated with corresponding changes in the activity of antioxidant enzymes.

Paracelsin E, a new peptaibol from Trichoderma saturnisporum.

The structure of paracelsin E, a new peptaibol from Trichoderma saturnisporum, has been determined primarily by fabms. The well-known paracelsins A, B, C, and D were also found in a culture of this organism.