Expression enhancement in brown adipose tissue of genes related to thermogenesis and mitochondrial dynamics after administration of pepsin egg white hydrolysate.

Nutritional compounds could be a safe and less expensive treatment for complications associated with obesity and metabolic syndrome (MetS). The aim of this study was to investigate the mechanism of action and the target tissues of a pepsin egg white hydrolysate (EWH) which had previously been demonstrated to improve some obesity-related disorders on a high-fat/high-glucose rat model. Wistar rats were used and divided into 3 groups: Control group (C), High-fat/high-glucose diet (MS) and high-fat/high-glucose diet + EWH (MSH). The rats were fed for 20 weeks and the EWH was administered from the 9th week. At the end of the study, white adipose tissue (WAT), brown adipose tissue (BAT) and muscle samples were collected for RT-qPCR analyses and immunohistochemistry. Our results showed a gene expression enhancement (2-fold basal level) in BAT of genes related to thermogenesis and mitochondrial dynamics. Mitochondrial DNA quantification and immunohistochemistry results also showed an increase of the mitochondrial content in this tissue. In conclusion, our results show the potential metabolic effect of this pepsin EWH by enhancing mitochondrial proliferation and gene expression related to thermogenesis in BAT. The EWH could be used as a functional food ingredient which is able to increase energy expenditure and counteract obesity-related MetS in a chronically obese society.

[The 5-word test in 37 depressed patients compared with 36 normal controls and 35 patients with mild Alzheimer's disease]. / Évaluation de la mémoire au moyen du Test des 5 mots chez 37 dépressifs comparés à 36 témoins et 35 patients ayant une forme légère de maladie d'Alzheimer.

OBJECTIVES: Patients with major depression (MD) express frequent memory complaints leading to consultations in memory clinics. The 5-word test (5WT) is a verbal memory test with semantic cueing, which has shown its sensitivity and its specificity in identifying patients with Alzheimer's disease (AD). Our objective was to evaluate memory performances of aged patients with MD compared with controls and AD patients. METHODS: Characteristics of the 5WT were investigated in a sample of 37 patients with MD (66.8±7.5 years) compared with 36 normal controls (67.3±6.8 years) and 35 mild AD patients (67.5±6.1 years). RESULTS: Duration of depression was 15.3±11.5 years. Memory complaints of MD patients were ancient (4.6±5.5 years) and severe (McNair memory questionnaire=47.6±20.7). The Total score of MD patients did not differ from controls but was greater than those of AD patients. Learning and Memory scores of MD patients were significantly lower than those of controls and significantly greater than those of AD patients. Forgetting rate between Learning and Memory scores was more important in AD (72.4%) than in controls (2.8%) and MD (13.6%). No intrusions were recorded in controls, three MD patients each made one intrusion, whereas 80% of AD patients made between one to six intrusions (mainly during cued delayed recall). Receiver operating characteristic curves determined the most significant cut-off scores of the Total score. It appeared easy to discriminate AD patients from controls (cut-off=9, sensitivity=94.3%, specificity=100%) or MD patients (cut-off=8, sensitivity=88.5%, specificity=89.2%) whereas it was more difficult to discriminate MD patients from controls (cut-off=10, specificity=88.9%, sensitivity=37.8%). DISCUSSION: MD patients had significant difficulties with the 5WT as compared to controls, without being of the magnitude of those observed in AD patients. CONCLUSION: The 5WT allows a reliable evaluation of memory in MD patients. The presence of true memory deficits with the 5WT could not be ascribed to depression but to other pathological conditions. Consequently, further memory testing should be conducted.

[The five-word test in three age-groups of mild Alzheimer's disease (60, 70 and 80 year-old patients): Utility of the Total Score, Total Weighted Score, Learning Score and Memory Score]. / Le Test des cinq mots dans les formes légères de maladie d'Alzheimer : comparaison du score total, du Score Total Pondéré, du Score d'apprentissage et du Score de mémoire dans trois classes d'âge (60 ans, 70 ans, 80 ans).

INTRODUCTION: The five-word test (5WT) is a serial verbal memory test with semantic cuing. It is proposed to rapidly evaluate memory of aging people and has previously shown its sensitivity and its specificity in identifying patients with AD. It measures the efficacy of free and cued recalls during a procedure of immediate and delayed recalls. METHODS: The 5WT was compared in a group of 202 normal subjects and a group of 302 mild AD patients (MMS of 20 or more) aged from 60 to 92 years, in three age classes (60 years, 70 years, 80 years). Nine scores were measured (Total Score, Total Weighted Score, Free Immediate Recall, Learning Score=total of Immediate Recalls, Free Delayed Recall, Memory Score=total of Delayed Recalls, Forgetting Rate, Percentage of Immediate Cuing, Percentage of Delayed Cuing) as well as the presence of intrusions. For each age class, Receiver Operating Characteristic curves determined the most significant cut-off scores. RESULTS: For each score of the 5WT, AD patients differed significantly from controls. The cut-off scores were not the same according to age. For the Total Score, the cut-off scores were 10 (60 years), 9 (70 years) and 8 (80 years), whereas the cut-off scores of the Total Weighted Score were 17 (60 years), 16 (70 years) and 14 (80 years). As suggested by Cowppli-Bony et al. (2005), the Total Weighted Score (which gives a higher coefficient to free recalls) was better than the Total Score for discriminating mild AD. The 5WT is useful to discriminate normal controls and mild AD patients. Normal aged subjects displayed good encoding, efficient stocking and consolidation (few forgetting, efficient cued recall), intrusions were rare. Mild AD patients were characterized by weak encoding of words and severe deficit for stocking and consolidation (important forgetting, impaired cued recall), they made numerous intrusions. This psychometric profile is characteristic of the amnestic hippocampal syndrome found in AD. CONCLUSION: The 5WT is a simple and reliable test for investigating memory in elderly people above 60 years old. According to age, different cut-offs are needed for the Total Score and the Total Weighted Score, the latter appearing more discriminating than the Total Score for the diagnosis of mild AD. It is also interesting to evaluate the presence of intrusions. Lastly, it is important to consider the forgetting rate (between Learning and Memory Scores) in order to confirm the presence of a hippocampal amnesia.

[Validation of the Rapid BAttery of Denomination (BARD) in 382 controls and 1004 patients of a memory clinic]. / Validation de la batterie rapide de dénomination (BARD) chez 382 témoins et 1004 patients d'une consultation mémoire.

INTRODUCTION: The Rapid BAttery of Denomination (BARD) is a short 10-item naming test derived from the 60-item Boston Naming Test. It is easily performed in less than 15 seconds by normal controls independently of age, gender and education (Croisile, 2005,2007,2008). Our aim was to evaluate the BARD in various conditions seen in a memory clinic. PATIENTS AND METHODS: The BARD was used in 382 normal subjects (165 men and 217 women, aged from 20 to 97 years) and 1004 patients attending a memory clinic. Three groups of 505 patients with Alzheimer's disease (AD) were compared: mild patients (n=402), moderate patients (n=84) and moderately severe patients (n=19). The BARD was also used in 499 patients with a Mini Mental Status (MMSE)>or=20: 173 patients with amnestic Mild Cognitive Impairment (aMCI), 56 patients with frontotemporal dementia (FTD), 41 patients with Lewy Body dementia (LBD), 36 patients with nonfluent primary progressive aphasia (NFPPA), 27 patients with semantic dementia (SD), 16 patients with posterior cortical atrophy (PCA), 150 patients with anxiety or depression (ADD). RESULTS: The performance of the patients was not affected by age, gender or education. aMCI had a score of 9.97+/-0.18, ADD a score of 9.97+/-0.2. A mild anomia was observed in three groups: mild AD (9.78+/-0.5), FTD (9.79+/-0.65) et LBD (9.98+/-0.16). A more pronounced anomia was present in moderate AD (9.10+/-1.06), moderately severe AD (8.05+/-1.27), PCA (8.12+/-3.28) and NFPPA (8.44+/-1.61). The anomia was severe in SD (5.85+/-2.46). The 10 items were perfectly named by 98 % of ADD, 96.53 % of aMCI, 82.09 % of mild AD, 87.5 % of FTD patients, 97.56 % of LBD patients, 68.75 % of PCA patients, but only 45.24 % moderate AD, 5.26 % of moderately severe AD, 27.78 % of NFPPA, and 3.7 % of SD. In the patients with MMS>or=20, Anova showed that the BARD scores of the ADD, aMCI, mild AD, FTD and LBD groups were significantly greater than the BARD scores of NFPPA, SD and PCA. PCA and NFPPA groups did not differ for BARD scores whereas they were significantly better than SD. A ROC curve comparing the 822 mild anomic patients (AD, FTD, LBD, aMCI, ADD) with the 79 more anomic patients (NFPPA, SD, PCA) showed that for a BARD score of 10, sensitivity was 72.2 %, specificity was 89.2 %, and 87.7 % of the patients were correctly classified. CONCLUSION: The BARD is a quick and useful tool for identifying naming disorders in a memory clinic. In patients with MMSE>or=20, making one error at the BARD is highly abnormal and significantly characteristic of cognitive disorders: the more frequent the errors are, the more probable is the presence of a visual agnosia (PCA), an aphasia (NFPPA), or a semantic disorder (SD).

[Standardization of the 5-word test in a group of 191 normal subjects aged 50 to 90 years]. / Etalonnage du test des cinq mots dans une population de sujets sains.

INTRODUCTION: The 5-word test (5WT) is a serial verbal memory test with semantic cuing. It allows to estimate cued learning, free recall, and cued recall of 5 words during immediate and delayed recalls (5 min later). The 5WT is proposed to rapidly evaluate memory of aging people. It has shown its sensitivity and its specificity in identifying patients with Alzheimer's disease (AD). OBSERVATIONS: We report the constitution of a sample of 191 French speaking normal subjects, aged from 50 to 90 years, in four age classes (50 years, 60 years, 70 years, 80 years) and three education levels. Total Score, Total Weighted Score, Free Delayed Recall, Delayed Recalls Total (memory score), and Free Recalls Total appear to be the best scores to appreciate the memory performance of the normal subjects. A Total Score of 10 was obtained in 74.9 p.cent of the subjects. No immediate nor delayed intrusions were recorded. In spite of the absence of an explicit consign, the recalled words were often ordered as they were in the list. CONCLUSION: The 5WT is a simple and reliable test for investigating memory in elderly people above 50 years old.

[Routine clinical diagnosis of primary progressive non-fluent aphasia]. / Diagnostic d'une aphasie progressive primaire non-fluente en pratique clinique courante.

It may be difficult to distinguish between a primary progressive aphasia at a very mild stage from the beginning of Alzheimer's disease (AD). However, this may be achieved by carrying out simple neuro-psychological tests. Nine non-fluent PPA (NFPPA) and 76 AD patients with comparable MMSE as well as 58 control subjects were evaluated using simple tests: MMSE, fluency, apraxia, naming, digital span, story memory, 5 words memory test. NFPPA patients had significantly impaired functions during the semantic category fluency and naming tests as compared to AD patients, whereas they showed a better delayed recall of the 5 words and story memory tests. As compared to AD, MMSE of NFPPA patients was also better in the time orientation and word recall sub-tests, although inferior in words repetition and language items. Thus, with comparable MMSE, NFPPA patients have more lexico-semantic difficulties, but a better delayed verbal memory than AD patients. These simple tests easily confirm the language impairment of NFPPA patients as opposed to the mnestic difficulties of AD, even at very early stages of these pathologies.

BPTI liquid-liquid phase separation monitored by light and small angle X-ray scattering.

In the field of protein crystallization, a better knowledge of the nucleation process is essential to control the nucleation rate, the growth and therefore the size and the quality of crystals. With that aim, it becomes clear that the important stage is the determination of the protein phase diagram. We highlighted and investigated the bovine pancreatic trypsin inhibitor (BPTI) binary liquid-liquid phase separation in 350 mM KSCN solutions as a function of temperature. We measured the low concentration part of the binodal curve using light scattering and optical microscopy. We show, from small angle X-ray scattering experiments, that the high concentrated phase sediments in the bottom of the capillary and we analysed the low concentrated phase in terms of monomers/decamers equilibrium.

Crystallization of a recombinant form of the complete sequence of human gamma-interferon: characterization by small-angle X-ray scattering, mass spectrometry and preliminary X-ray diffraction studies.

The crystallization conditions of a recombinant form of the complete sequence of human gamma-interferon, designated r-hu IFN-gamma (RU 42369), have been determined after studying the behaviour of this protein in solution by small-angle X-ray scattering (SAXS) as a function of pH and salt type. IFN-gamma is difficult to crystallize without truncating at least the last five amino acids of the C-terminus; the SAXS results suggest viable crystallization conditions that led to crystals of r-hu IFN-gamma suitable for X-ray diffraction analysis. The crystals were grown in the presence of ammonium sulfate using vapour-diffusion techniques. The crystals, which diffract to 5 A resolution at best, belong to the primitive tetragonal space group P42(1)2 and have unit-cell parameters a = b = 123.4, c = 93.4 A. The protein contained in these crystals was analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), which verified the presence of the complete amino-acid sequence of r-hu IFN-gamma.

The decameric structure of bovine pancreatic trypsin inhibitor (BPTI) crystallized from thiocyanate at 2.7 A resolution.

The structure of a monoclinic form of bovine pancreatic trypsin inhibitor (BPTI) crystallized from a thiocyanate solution has been determined and refined at 2.7 A resolution. The space group is P21 with a = 71.56, b = 73.83, c = 64.47 A, beta = 93.9 degrees and Z = 20. The ten independent molecules were located by a multi-body molecular-replacement search as developed in the AMoRe program, starting from a single monomer model (PDB code: 6PTI). The molecular arrangement of the subunits is a decamer resulting from the combination of two orthogonal fivefold and twofold non-crystallographic axes. This builds a globular micelle-like particle which minimizes hydrophobic interactions with the solvent. The refinement was conducted with non-crystallographic symmetry constraints up to a final residual of R = 0.20 (Rfree= 0.26). The root-mean-square deviations from ideal geometry were 0.015 A and 1.6 degrees on bond distances and bond angles, respectively. Several sites for thiocyanate ions were analyzed.

Protein crystals orientation in a magnetic field.

Nucleation and crystal growth of hen egg-white lysozyme, bovine pancreatic trypsin inhibitor and porcine pancreatic alpha-amylase were carried out in the presence of a magnetic field of 1.25 T produced by small permanent magnets. Crystals were oriented in the magnetic field, except when heterogeneous nucleation occurred. The orientation of protein crystals in the presence of a magnetic field can be attributed to the anisotropic diamagnetic susceptibility of proteins resulting from the large anisotropy of the alpha-helices due to the axial alignment of the peptide bonds.

Pancreatic lithostathine as a calcite habit modifier.

Most biological fluids are supersaturated with calcium salts. A mechanism controlling crystal growth is therefore necessary to prevent excessive precipitation and development of a lithiasis. In pancreatic juice, calcite precipitation is prevented by lithostathine, a glycoprotein that inhibits calcite crystal growth. We describe here the interaction of lithostathine with calcite crystals. Without lithostathine, calcite crystals grew as rhombohedra showing six (104) faces. At low concentration (1 microM), lithostathine already altered crystal growth by generating new (110) faces. At physiological concentrations (3-10 microM), adsorption resulted in a transition from rhombohedral to sub-cubic habits. Immunochemical localization demonstrated that, although all (104) faces are equivalent, lithostathine binding was restricted to the face edges distal to the c axis. Scanning electron microscopy showed that, at the site of lithostathine binding, spreading of new CaCO3 layers during crystal growth was arrested before reaching the crystal diad axis-bearing edges. The successive kinks generated during crystal growth formed the new, striated (110)faces. Similar modifications were observed with the N-terminal undecapeptide of lithostathine that bears the inhibitory activity. With 100 microM lithostathine, (110) faces could reach the c axis outcrop of the former rhombohedron, resulting in an olive-shaped crystal. Finally, the number of crystals increased and their average size decreased when lithostathine concentration increased from 0.1 to 100 microM. Decreased Ca2+ concentration during crystal growth was delayed in the presence of lithostathine. It was concluded that lithostathine controls lithogenesis 1) by triggering germination of numerous calcite crystals and 2) by inhibiting the rate of Ca2+ ion apposition on the nuclei and therefore interfering with the apposition of new layers on calcite. Formation of smaller crystals, whose elimination is easier, is thereby favored.

Calcium carbonate crystals promote calcium oxalate crystallization by heterogeneous or epitaxial nucleation: possible involvement in the control of urinary lithogenesis.

A large proportion of urinary stones have calcium oxalate (CaOx) as the major mineral phase. In these stones, CaOx is generally associated with minor amounts of other calcium salts. Several reports showing the presence of calcium carbonate (CaCO3) and calcium phosphate in renal stones suggested that crystals of those salts might be present in the early steps of stone formation. Such crystals might therefore promote CaOx crystallization from supersaturated urine by providing an appropriate substrate for heterogeneous nucleation. That possibility was investigated by seeding a metastable solution of 45Ca oxalate with vaterite or calcite crystallites. Accretion of CaOx was monitored by 45Ca incorporation. We showed that (1) seeds of vaterite (the hexagonal polymorph of CaCO3) and calcite (the rhomboedric form) could initiate calcium oxalate crystal growth; (2) in the presence of lithostathine, an inhibitor of CaCO3 crystal growth, such accretion was not observed. In addition, scanning electron microscopy demonstrated that growth occurred by epitaxy onto calcite seeds whereas no special orientation was observed onto vaterite. It was concluded that calcium carbonate crystals promote crystallization of calcium oxalate and that inhibitors controlling calcium carbonate crystal formation in Henle's loop might play an important role in the prevention of calcium oxalate stone formation.

Crystal structure of pig pancreatic alpha-amylase isoenzyme II, in complex with the carbohydrate inhibitor acarbose.

Two different crystal forms of pig pancreatic alpha-amylase isoenzyme II (PPAII), free and complexed to a carbohydrate inhibitor (acarbose), have been compared together and to previously reported structures of PPAI. A crystal form obtained at 4 degrees C, containing nearly 72% solvent, made it possible to obtain a new complex with acarbose, different from a previous one obtained at 20 degrees C [Qian, M., Buisson, G., Duée, E., Haser, H. & Payan, F. (1994) Biochemistry 33, 6284-6294]. In the present form, six contiguous subsites of the enzyme active site are occupied by the carbohydrate ligand; the structural data indicate that the binding site is capable of holding more than the five glucose units of the scheme proposed through kinetic studies. A monosaccharide ring bridging two protein molecules related by the crystal packing is located on the surface, at a distance of 2.0 nm from the reducing end of the inhibitor ligand; the symmetry-related glucose ring in the crystal lattice is found 1.5 nm away from the non-reducing end of the inhibitor ligand.

Influence of polydispersity on protein crystallization: a quasi-elastic light-scattering study applied to alpha-amylase.

The early stages of the crystallization process of porcine pancreatic alpha-amylase were investigated by quasi-elastic light scattering. It is shown that at 288 and 293 K the diffusion coefficient does not monotonically change with increasing protein concentration but passes through a maximum at 10 mg ml(-1). In supersaturated solutions, prior to nucleation, the protein is strictly monodisperse. Nucleation induces the formation of aggregates and a polydispersity of, for example, 18% for an initial supersaturation C/C(e) = 5.8. Monodispersity is restored after the nuclei have grown and partially consumed the solute. On the other hand, polydispersity increases up to 20% at 298 K if the protein concentration decreases to 3-4 mg ml(-1), values at which the solutions are under-saturated. When the protein concentration exceeds 5-6 mg ml(-1) the protein becomes monodisperse again. These results, confirmed by those of another system we are studying (bovine pancreatic trypsin inhibitor), are at variance with the statements that supersaturation is always at the origin of aggregation and polydispersity, and that in undersaturated solutions the diffusion coefficient should remain constant for obtaining crystals once the solutions are supersaturated.

Crystal structure of toxin II from the scorpion Androctonus australis Hector refined at 1.3 A resolution.

The crystal structure of toxin II from the scorpion Androctonus australis Hector has been refined at 1.3 A resolution using restrained least-squares methods. The final R-factor is 0.148 for the 13,619 reflections between 7.0 A and 1.3 A resolution with F > 2.5 sigma (F) and the bond length standard deviation from ideality is 0.017 A. Although minor changes have been introduced relative to the model previously refined at 1.8 A resolution, the use of higher-resolution data has allowed the modelling of some discrete disorder. Thus, three residues (including a disulphide bridge) have been built with multiple conformations. Occupancies were refined for the 106 solvent molecules included in the model, nine of them with explicit multiple sites. There is well-defined electron density for some of the protein hydrogen atoms in the final difference Fourier map. A detailed description of the toxin structure is presented, along with a comparison with the high-resolution structure of the related variant-3 scorpion toxin.

Crystallization of barley malt alpha-amylases and preliminary x-ray diffraction studies of the high pI isozyme, alpha-amylase 2.

alpha-Amylase isozymes 1 and 2 isolated from germinated barley seeds have been crystallized by the hanging- or sitting-drop vapor diffusion technique. Crystals of alpha-amylase 2 suitable for x-ray diffraction analysis were grown at pH 6.7 and 22 degrees C from a solution of 1 mM calcium chloride, 10 mM MES, and 16% saturated ammonium sulfate. The space group is trigonal P3121 (or P3221) with unit cell dimensions a = b = 135.20 A, c = 79.63 A, and probably two molecules per asymmetric unit.

Preliminary crystallographic data on a ferredoxin from Desulfovibrio desulfuricans (Norway strain).

The ferredoxin (Fd I) (Mr2 X 6000, one (4 Fe-4 S) cluster per subunit) from the sulphate-reducing bacteria Desulfovibrio desulfuricans Norway 4 has been crystallized. The space group is P4(2)32 with a = 71.8 A. The two monomers of the molecule are probably related by a dyad axis.

Crystal structure and electron transfer properties of cytochrome c3.

The crystal structure of cytochrome c3 from the sulfate-reducing bacteria Desulfovibrio desulfuricans, Norway strain, has been determined through the fitting of the recently completed primary structure to a 2.5 A resolution electron density map. The phase calculations were based on three mercurial derivatives; anomalous scattering data were used to refine the four heme iron positions. A preliminary refinement of the molecular model has led to a conventional crystallographic R factor of 34%. Cytochrome c3 is folded in two structural domains with one heme in each, the two other heme moieties lying in a large groove dividing the molecule. The core of the protein is the compact four-heme cluster which presents a relatively high degree of solvent exposure. The structural pattern of redox centers suggests that electron transfer might occur through direct contacts between some of the heme groups, via the overlapping system of pi oribitals or via intervening amino acid side chains or both.

Pancreatic colipase: crystallographic and biochemical aspects.

A detailed study of the crystallization of hog and horse colipases has been undertaken. Several crystallographic varieties have been obtained and a 0.3-nm resolution structure determination is actually in progress. The sequence of the A form of horse colipase (one methionine) is given. From spectrophotometric experiments and sequence comparisons, the involvement of the aromatic residue in position 52 in the micelle binding site has been demonstrated.

Pancreatic basal secretion in alcohol-fed and normal dogs.

Biochemical, histological, and crystallographic studies were carried out on basal pancreatic secretion of 4 dogs fed alcohol for 12-15 months and 11 control dogs. The results in alcohol-fed dogs when compared to normals showed that: (1) protein concentration was higher, (2) fluid was decreased; (3) conductivity was decreased leading to differences in ionic distribution: Cl- and H+ ion concentrations increased, HCO3 concentrations and output decreased, total Ca, Mg, and Zn soluble in the juice did not change and therefore Ca, Mg, and Zn to protein ratios decreased. In the basal secretion of alcoholic dogs, numerous plugs were found of which three components were identified: (1) cells mostly of ductal origin; (2) calcium already crystallized; (3) protein material in the center of these plugs. Thus a change in Donan equilibrium led to modifications of protein and calcium solubilities with formation of precipitates. These findings are relevant to the study of chronic pancreatitis due to alcohol consumption.