Relevance of the serial position effect in the differential diagnosis of mild cognitive impairment, Alzheimer-type dementia, and normal ageing.

UNLABELLED: Serial position effects are observed when a person memorises a series of words exceeding his or her attention span. Cognitively normal individuals recall words at the beginning and end of the list more frequently than those in the middle, which reflects the way that short- and long-term episodic memory works. OBJECTIVE: To study the serial position effect in patients with mild cognitive impairment (MCI) compared to subjects with Alzheimer-type dementia (AD) or normal ageing (NA). METHODS: 30 AD, 25 MCI and 20 NA subjects underwent neurological and neuropsychological assessment. The Rey Auditory Verbal Learning Test (RAVLT) was used to study primacy, middle, and recency effects and delayed recall for each group. RESULTS: The general memory pattern of MCI subjects was very similar to that of AD subjects, and was characterised by reduced learning capacity, rapid forgetfulness and clear recency effect in learning. With regard to delayed recall, however, there were differences in performance; MCI subjects' ability to recall words at the beginning and middle of the list was similar to that of normal subjects, while their memory of words at the end of the list was poor, as in AD subjects. CONCLUSIONS: RAVLT is a tool permitting us to distinguish between MCI and NA subjects. The recency index for the delayed recall task is a valid indicator for distinguishing between MCI patients and patients with normal ageing.

Characteristics of an environmental strain, Enterococcus faecalis CECT7121, and its effects as additive on craft dry-fermented sausages.

Lactic acid bacteria are the most adequate microorganisms for natural preservation of food. In the present work, the strain of Enterococcus faecalis CECT7121 was employed in the manufacture of craft dry-fermented sausages and its performance as a biopreservative was analysed. This strain is devoid of the genes for haemolysin and gelatinase and does not produce biogenic amines. It is sensitive to almost all the antibiotics tested and opsonophagocytic assays showed that it is devoid of a capsule. This strain had a high LD50 (10(11)CFU ml(-1)) in mice. No statistical differences were found between control and sausages inoculated with E. faecalis CECT7121 regarding the production of lactic acid, pH variation over time, reaching a minimum pH value of 5.1, and sensory analysis in both series. Sausages inoculated with E. faecalis CECT7121 had lower viable counts of Enterobacteriaceae, Staphylococcus aureus and other Gram-positive cocci at the end of fermentation and 7 days and no viable enterobacteria and S. aureus were recovered at the end of drying. E. faecalis CECT7121 did not affect the growth of Lactobacillus spp. but it displaced the autochthonous populations of enterococci. E. faecalis CECT7121 was recovered in each time point as assessed by its inhibitory activity on Listeria monocytogenes and S. aureus. These results would indicate that the addition of E. faecalis CECT7121 during the manufacture of craft dry-fermented sausages offers an interesting alternative for biopreservation.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 A resolution.

BACKGROUND: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. RESULTS: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 A resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. CONCLUSIONS: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

The allosteric transition of glucosamine-6-phosphate deaminase: the structure of the T state at 2.3 A resolution.

BACKGROUND: The allosteric hexameric enzyme glucosamine-6-phosphate deaminase from Escherichia coli catalyses the regulatory step of N-acetylglucosamine catabolism, which consists of the isomerisation and deamination of glucosamine 6-phosphate (GlcN6P) to form fructose 6-phosphate (Fru6P) and ammonia. The reversibility of the catalysis and its rapid-equilibrium random kinetic mechanism, among other properties, make this enzyme a good model for studying allosteric processes. RESULTS: Here we present the structure of P6(3)22 crystals, obtained in sodium acetate, of GlcN6P deaminase in its ligand-free T state. These crystals are very sensitive to X-ray radiation and have a high (78%) solvent content. The activesite lid (residues 162-185) is highly disordered in the T conformer; this may contribute significantly to the free-energy change of the whole allosteric transition. Comparison of the structure with the crystallographic coordinates of the R conformer (Brookhaven Protein Data Bank entry 1 dea) allows us to describe the geometrical changes associated with the allosteric transition as the movement of two rigid entities within each monomer. The active site, located in a deep cleft between these two rigid entities, presents a more open geometry in the T conformer than in the R conformer. CONCLUSIONS: The differences in active-site geometry are related to alterations in the substrate-binding properties associated with the allosteric transition. The rigid nature of the two mobile structural units of each monomer seems to be essential in order to explain the observed kinetics of the deaminase hexamer. The triggers for both the homotropic and heterotropic allosteric transitions are discussed and particular residues are assigned to these functions. A structural basis for an entropic term in the allosteric transition is an interesting new feature that emerges from this study.

Glucosamine-6-phosphate deaminase from beef kidney is an allosteric system of the V-type.

The enzyme glucosamine-6-phosphate deaminase from beef kidney has been purified to homogeneity by allosteric-site affinity chromatography. Its amino acid composition and the N-terminal sequence (1-42), were obtained. The amino acid sequence of this segment is essentially identical to the corresponding regions of the human and hamster glucosamine-6-phosphate deaminases. The beef enzyme is a hexamer of 32.5 kDa subunits; this is nearly 2.5 kDa higher than the molecular mass of the homologous enzyme from Escherichia coli. Beef kidney deaminase exhibits a notable difference from the bacterial enzyme in its allosteric activation by N-acetylglucosamine 6-phosphate This metabolite, which is also is the allosteric activator of the bacterial glucosamine-6-phosphate deaminase, activates the enzyme by increasing its kcat without any change in the Km values for glucosamine 6-phosphate, over a wide range of activator concentration. This observation places beef kidney deaminase in the class of V-type allosteric systems.

Zinc binding and its trapping by allosteric transition in glucosamine-6-phosphate deaminase from Escherichia coli.

Glucosamine-6-phosphate isomerase deaminase from Escherichia coli, a typical allosteric enzyme, becomes less cooperative and 50% inhibited when treated with zinc. This metal cation behaving as a tight-bound and slow partial inhibitor. Modification of a pair of vicinal reactive thiols with some sulfhydryl reagents mimics this effect. On the other hand, sulfhydryl reactivity disappears in the presence of saturating concentrations of Zn2+, which does not modify the kinetics of S-methylated enzyme, a finding that indicates that vicinal thiols are an essential part of the zinc-binding site. Allosteric activation of the deaminase causes trapping of the metal, which cannot be released by dialysis against a buffer containing EDTA. Cadmium and nickel(II) cations also produce a similar effect.

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy.

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.

Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli.

Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity. The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-epsilon-amino-n- caproyl -D-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P. The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 +/- 300 Da; the isoelectric point was 6.0-6.1 and the sedimentation constant 9.0 S. The amino-acid composition of the enzyme was determined and a value for E1%275 of 4.55 was calculated. The molecular activity was 1800 s-1 for the deamination reaction and 455 s-1 for the reaction of GlcN6 P formation. A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7). Ammonia behaved as a Michaelian substrate. Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on Km for ammonia. The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the Km values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.

On the role of the N-terminal group in the allosteric function of glucosamine-6-phosphate deaminase from Escherichia coli.

Glucosamine-6-phosphate deaminase (EC 3.5.99.6) from Escherichia coli is an allosteric enzyme of the K-type, activated by N-acetylglucosamine 6-phosphate. It is a homohexamer and has six allosteric sites located in clefts between the subunits. The amino acid side-chains in the allosteric site involved in phosphate binding are Arg158, Lys160 and Ser151 from one subunit and the N-terminal amino group from the facing polypeptide chain. To study the functional role of the terminal amino group, we utilized a specific non-enzymic transamination reaction, and we further reduced the product with borohydride, to obtain the corresponding enzyme with a terminal hydroxy group. Several experimental controls were performed to assess the procedure, including reconditioning of the enzyme samples by refolding chromatography. Allosteric activation by N-acetylglucosamine 6-phosphate became of the K-V mixed type in the transaminated protein. Its kinetic study suggests that the allosteric equilibrium for this modified enzyme is displaced to the R state, with the consequent loss of co-operativity. The deaminase with a terminal hydroxy acid, obtained by reducing the transaminated enzyme, showed significant recovery of the catalytic activity and its allosteric activation pattern became similar to that found for the unmodified enzyme. It had lost, however, the pH-dependence of homotropic co-operativity shown by the unmodified deaminase in the pH range 6-8. These results show that the terminal amino group plays a part in the co-operativity of the enzyme and, more importantly, indicate that the loss of this co- operativity at low pH is due to the hydronation of this amino group.

Crystallization and preliminary crystallographic studies of glucosamine-6-phosphate deaminase from Escherichia coli K12.

Hexameric glucosamine-6-phosphate deaminase from Escherichia coli has been crystallized isomorphously with both phosphate and ammonium sulphate as precipitants, over a wide pH range (6.0 to 9.0). The crystals belong to space group R32 and the cell parameters in the hexagonal setting are a = b = 125.9 A and c = 223.2 A. A complete native data set was collected to 2.1 A resolution. Self-rotation function studies suggest that the hexamers sit on the 3-fold axis and have point group symmetry 32, with a non-crystallographic dyad relating two monomers linked by an interchain disulfide bridge. A possible packing for the unit cell is proposed.

[Validity of the Spanish version of the Addenbrooke's Cognitive Examination for the diagnosis of dementia and to differentiate Alzheimer's disease and frontotemporal dementia]. / El Addenbrooke's Cognitive Examination en español para el diagnóstico de demencia y para la diferenciación entre enfermedad de Alzheimer y demencia frontotemporal.

INTRODUCTION: The Addenbrooke's Cognitive Examination (ACE) is a brief bedside test battery to detect mild dementia and differentiate frontotemporal dementia (FTD) from Alzheimer's disease (AD). AIM: To validate the ACE in Spanish. PATIENTS AND METHODS: The study evaluated the Spanish version of ACE on 128 subjects consisting in two groups a patient group (n = 76) and a control subjects group (n = 52). The patient group was divided in AD (n = 54) based on the NINCDS-ADRDA criteria and FTD (n = 22) based on the Lund y Manchester criteria. All patients underwent clinical, neuropsychological, radiologic (MRI, CT, and SPECT), and laboratory evaluations. Group's differences were evaluated using ANOVA. The internal consistency of the Spanish version of the ACE was measured using the Cronbach's alpha coefficient. The discriminative capability of the Spanish version of the ACE was examined by the receiver operating characteristic (ROC) analysis. RESULTS: The cut-off score of 86 showed a sensitivity of 92% (CI 95% = 83.6-97.0) and a specificity of 96.2% (CI 95% = 86.8-99.4). The ROC curve showed higher sensitivity and specificity of the ACE than the Mini-Mental State Examination in discriminating the dementia and control group. The VLOM ratio (verbal fluency + language)/(orientation + memory) of < 1.82 discriminated for FTD and > 4.87 discriminated for AD. CONCLUSION: The Spanish version of ACE is a brief and reliable instrument for early detection of dementia in highly educated people and offers a simple objective index to differentiate AD and FTD. More studies in less educated people are warranted.

Determination of the inducers of Fc (FcRI) and C3 (C3RI) receptors on myeloid cells in several media from human and mouse origin, and the identification of the macrophage as the cell that produces these factors.

The presence of factors responsible for the induction of Fc receptors (FcRI) and C3 receptors (C3RI) on mouse myeloid cells was determined in postendotoxin serum and in media conditioned by postendotoxin lungs, peritoneal macrophages, peritoneal granulocytes, and lymphocytes from either the lymph nodes, the thymus, or the spleen, FcRI and C3RI were found only in postendotoxin serum and in media conditioned by either postendotoxin lungs or peritoneal macrophages. The apparent molecular weights obtained by molecular sieving chromatography in Sephadex G-100 were 10,500 daltons for FcRI and 35,000 daltons for C3RI. These results suggest the existence of several molecules responsible for induction of Fc and C3 receptors. Under our experimental conditions, the macrophage was the only blood cell capable of FcRI and C3RI production, thus hinting at a possible feedback regulatory mechanism. In order to determine whether both inducing factors are also present in normal individuals, human serum and urine were used. FcRI and C3RI were both found in human serum, while only the FcRI factor was detected in urine. These results indicate that the normal human being produces FcRI and C3RI, and that probably the C3RI does not pass through the kidney barrier. The mechanism of action of these factors can be studied by using mouse cells.

Influence of the level of arsenic (As) exposure and the presence of T860C polymorphism in human As urinary metabolic profile.

The influence, on arsenic (As) urinary metabolic profile, of the level of As exposure was evaluated on chronic-exposed inhabitants of several locations of the Chaco-Pampean Plains in Argentina. Urinary As (UAs) was quantified as a measure of the level of exposure. The metabolic profile of UAs (inorganic As, monomethylarsonic acid, and dimethylarsinic acid) was also evaluated. The presence of T860C polymorphism on the arsenite methyltransferase encoding gene was investigated by desquamation of buccal cells. UAs showed a wide range of levels (from 18 µg/g to 4103 µg/g) of creatinine. A clear influence of age, gender, level of As exposure, and the presence of T860C polymorphism was observed on As metabolic profile. The influence of the level of exposure showed to be different between individuals carrying the wild type (WT) and the heterozygous (H) genotypes. Metabolic profile of individuals carrying the WT genotype seemed to be influenced by the level of exposure, while individuals with the H genotype did not. It is concluded that the level of As exposure seemed to have a significant influence on urinary metabolic profile of individuals carrying the WT genotype. In contrast, individuals carrying the H genotype seemed not to be affected the same way by increasing the As exposure level.

Interaction of Leishmania mexicana promastigotes with the plasminogen-plasmin system.

The binding of human plasminogen and plasmin to the promastigote form of Leishmania mexicana was investigated. L. mexicana was capable to bind both molecules, the binding being inhibited by epsilon-aminocaproic acid. Scatchard plot analysis revealed a dissociation constant (Kd) value of 2.4+/-0.8 microM and 0.9+/-0.1 x 10(4) binding sites per cell for plasminogen and a Kd value of 1.2+/-0.4 microM and 1.6+/-0.2 x 10(5) binding sites per cell for plasmin. C-terminal lysine residues are involved in plasminogen binding to cells, since carboxypeptidase B treatment reduced this binding by 34%. Ligand blotting analysis showed a group of proteins, with molecular masses between 105 and 115 kDa, capable to interact with plasminogen. Zymogram analysis showed that the protease activity acquired by L. mexicana, due to the interaction with either plasminogen or plasmin, comprises an important fraction of the total protease activity at pH 7.7. Plasminogen activation by tissue-type plasminogen activator (t-PA) was enhanced by the presence of L. mexicana promastigotes. These results raise the question whether the interaction of L. mexicana with components of the fibrinolytic system is involved in the virulence of the parasite.

99mTc-ENS: a new radiopharmaceutical for aerosol lung scintigraphy. Comparison between different freeze-dried formulations.

UNLABELLED: Exogenous natural surfactant (ENS) labeled with 99mTc(99mTc-ENS) is a new radiopharmaceutical for pulmonary aerosol scintigraphy. In this study, different freeze-dried formulations were evaluated to develop a suitable and long-storage method for the ENS, the nonradioactive precursor of this radiopharmaceutical. METHODS: Two freeze-dried formulations were evaluated: the sterile ENS suspension-stannous chloride altogether lyophilized (chlorlioENS) and the lyophilized sterile ENS suspension with the addition of stannous chloride as a solid drug (lioENS). These precursors were stored at room temperature for 3 mo and then labeled with 99mTc. For comparative purposes, the sterile ENS suspension with the addition of stannous chloride labeled with 99mTc(99mTc-chlorENS) was also studied. The quality controls for each radiopharmaceutical were performed by an ascending paper chromatography to determine the labeling yield percentages. The study was performed in 30 female Sprague Dawley rats, which inhaled each radiopharmaceutical by nebulization. Twenty-five minutes after the aerosol inhalation, the animals were killed to extract their organs and measure their activity in a gamma spectrometer. The data are given as the percentage of activity concentration (C%) for each organ. RESULTS: The physicochemical properties of lioENS were adequate for a freeze-dried product. The labeling yields for 99mTc-lioENS and for 99mTc-chlorENS were always greater than 95% even after nebulization. The results of the biologic distribution studies showed that the activity concentration found in lungs for these radiopharmaceuticals were 95.7% +/- 2.6% and 96.7% +/- 2.6% respectively, results that do not differ statistically. On the other hand, the activity concentration found in lungs for the 99mTc-chlorlioENS (31.3% +/- 11.1%) and its labeling yield percentages (<10%) are statistically different (P < 0.05) from the results obtained with the two radiopharmaceuticals mentioned above. CONCLUSION: Taking into account the lioENS physicochemical properties, its long shelf life and that 99mTc-lioENS shows the same radiochemical and radiopharmacological behavior of the 99mTc-chlorENS, it can be concluded that the 99mTc-lioENS can be used for aerosol lung scintigraphy.

Aproteic diet decreases hypothalamic catecholamine turnover in adult male rats.

Previous reports indicate that malnutrition reduces reproductive functions. We have demonstrated that protein deprivation in the diet also causes reproductive dysfunction by reducing hypothalamic GnRH secretion. Noradrenaline and nitric oxide are modulators of GnRH secretion. Noradrenaline stimulates GnRH secretion and nitric oxide inhibits catecholamine release. This work studies the hypothalamic catecholaminergic and nitrergic neuron activity in Wistar adult male rats fed on an aproteic diet (AP) during 21 days; this treatment was started when rats were 70 days old. Our first experiment studied catecholamine turnover rate after inhibition of tyrosine hydroxylase activity by injecting (i.p.) 400 mg/kg alpha-methyl-p-tyrosine. Our second experiment studied in vitro hypothalamic nitric oxide synthase (NOS) activity in animals under the same diet. AP diet significantly decreased both noradrenaline (P<0.05) and dopamine (P<0.05) hypothalamic turnover rate. Noradrenaline turnover in cerebral cortex was not altered by the aproteic diet. However, hypothalamic NOS activity was not affected in animals fed on an AP diet. These results indicate that the lack of protein in the diet reduces catecholaminergic neuron activity in adult male rats by a NO-independent mechanism, thus suggesting that a decrease in noradrenergic activity may be involved in the reduction of GnRH secretion induced by an AP diet.

MIN 14C UBT: a combination of gastric basal transit and 14C-urea breath test for the detection of Helicobacter pylori infection in human beings.

The purpose of this work is to demonstrate that the 14C-urea breath test (UBT) performed at different times combined with the study of the gastric basal transit, which evaluates the intragastric displacement of a labeled solution under fasting conditions, has the advantage of being representative of the whole stomach surface and constitutes a non-aggressive test for the detection of H. pylori. This test, which has been called MIN 14C UBT, is a modification of the conventional 14C UBT in which low volumes of a solution of 14C-urea together with 99mTc-sulfur colloid are administered. The 99mTc-sulfur colloid is not absorbed in the gastrointestinal tract and has the great advantage of allowing the "visualization" of the transit of the 14C-urea within the gastrointestinal tract. This modification allows the simultaneous determination of the production of the 14CO2 and the place where this process occurs. The results show that there is a good correlation between the images obtained and the breath samples collected. We found that this test has a sensitivity of 98% and a specificity of 96% for H. pylori detection.

A thymus factor influences the in vitro testosterone secretion of Leydig cells in the rat.

Thymus extracts obtained from 15-day-old rats were fractionated through molecular sieve chromatography, and the fractions assayed in vitro by changes produced in the testosterone secretion of Leydig cells obtained from adult rat testes. Fractions corresponding to 27-28000 mol wt of the thymus extract diminish the testosterone secretion of Leydig cells stimulated with hCG. No changes in the basal testosterone secretion were produced by the presence of the thymus fractions. The inhibitory effect is dose related and persists during 180 min of incubation. Fractions of the same mol wt obtained from liver, heart and spleen do not modify the testosterone secretion of Leydig cells. The inhibitory activity of the thymus factor disappears after heat or trypsin treatment. Further fractioning in preparative flat bed electrofocusing makes manifest that the inhibitory activity is focused at pH 4.7. The data demonstrate the existence in rat thymus of a factor, probably of protein nature, which modifies the in vitro hCG response of a testis cell suspension.

Cytotoxic effect of Argentine medicinal plant extracts on human hepatocellular carcinoma cell line.

Methanolic extracts from Achyrocline satureioides (Dc.) Lam, Aristolochia macroura Gomez, Lithraea molleoides (Vell.) Engl., Schinus molle L., unlike those from Celtis spinosa Spreng, Chenopodium ambrosioides L., Petiveria alliacea L., and Plantago major L. showed cytotoxic activity against a human hepatocellular carcinoma cell line, Hep G2. Schinus molle L. was the most active (IC50=50+/-7 microg/ml). These results call for further studies of these extracts.

[Leucocyte tartrate-resistant acid phosphatase as marker for the transition of monocyte to macrophage]. / Fosfatasa ácida leucocitaria tartrato resistente como marcador de la transformación del monocito en macrófago.

The presence of the 5 degrees isoenzyme of leukocyte tratrate-resistant acid phosphatase (FATRE) was investigated in human peripheral blood monocytes in 32 samples: 26 normal, 4 thrombocytopenia, 1 anemia and 1 hairy cell leukemia. The Cobe Spectra Version 4 cell separador was used for 3 samples while the others were obtained by centrifugation with or without latex particles in order to study macrophages and monocytes, respectively. Using a Sigma Kit for both total acid phosphatase and FATRE reactions, the presence of two monocyte populations was detected, one slightly positive and the other negative for FATRE. Upon the addition of latex particles, the monocytes were transformed into intensely FATRE positive macrophages. It can be concluded that FATRE must play an important role in macrophage function and consequently in human cell immunity.