Juan M. García-Segura

Proton Magnetic Resonance Spectroscopy and Magnetoencephalographic Estimation of Delta Dipole Density: A Combination of Techniques That May Contribute to the Diagnosis of Alzheimer's Disease

Whole-head magnetoencephalographic recordings were obtained from 10 patients with Alzheimer’s disease (AD) and 10 healthy controls in a resting position. Spectroscopic examinations were performed by means of a 1.5-tesla whole-body scanner in the temporoparietal regions of both hemispheres. The relationship between 1H-MRS-based and magnetoencephalography (MEG)-based measures and their conjoined capability to improve the diagnosis of AD were investigated in this study. Logistic regression analyses were performed. Three separated logistic models were calculated for 1H-MRS-based metabolites, low-frequency magnetic activity, and the combination of both measures. A combined myoinositol/N-acetyl aspartate (mI/NAA)-delta dipole density (DD) model predicted the diagnosis with 90% sensitivity and 100% specificity. Additionally, the combination of temporoparietal mI/NAA and delta DD values explained the variability of individuals’ cognitive status. The results support the notion that a multidisciplinary approach may improve the understanding and diagnosis of AD.

Evidence of Biochemical and Biomagnetic Interactions in Alzheimer’s Disease: An MEG and MR Spectroscopy Study

Background: Several neuroimaging studies have shown reliable differences between Alzheimer’s disease (AD) patients and age-matched controls. However, few studies have demonstrated the interactions between neuroimaging methods for the diagnoses of AD. Objective: In this study, we try to elucidate the complementary nature of magnetoencephalography (MEG) and magnetic resonance spectroscopy (MRS) examinations in the assessmentof AD. Methods: Ten patients fulfilling the NINCDS-ADRDA criteria of probable AD, and 10 elderly individuals with no history of neurological or psychiatric illness serving as age-matched controls participated in the study. All patients and controls received an MRS, MEG and neuropsychological assessment. MEG data were obtained in the context of a working memory task, previously utilized in a similar sample of patients. Results: The AD group showed a reduced number of activity sources over left temporoparietal areas during the late portion of the evoked magnetic field (between 400–800 ms), as well as a bilateral temporoparietal increase in creatine and myoinositol concentrations, and in the myoinositol/N-acetyl-aspartate ratio. The combination of the variables ‘number of dipoles during the late portion of the evoked magnetic field’ and ‘myoinositol/N-acetyl-aspartate ratio’ accounted for 65% of the variance of the Mini Mental State Examination scores. Conclusions: These results highlight the importance of assessing the complex brain pathology underlying AD by utilizing multiple brain examination modalities in a coordinate approach.

Thiol-Disulfide Exchange of Ribonuclease Inhibitor Bound to Ribonuclease A EVIDENCE OF ACTIVE INHIBITOR-BOUND RIBONUCLEASE

Ribonuclease Inhibitor (RI) has been purified from pig testis. It contains 30 half-cystines whose oxidation affects its ability to bind and inhibit ribonuclease (RNase). By N-terminal sequence analyses testis RI showed to be identical to that from porcine liver, for which a characteristic all-or-none type of SH-oxidation by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported (Fominaya, J. M., and Hofsteenge, J.(1992) J. Biol. Chem. 257, 24655-24660). Under comparable reaction conditions, testis RI bound to RNase A did not exhibit this particular type of oxidation; instead, bound RI got intermediate oxidation degrees (up to 14 thiols oxidized per RI moiety) without dissociating from RNase. Moreover, RNase bound to partially oxidized RI was able to express some (15%) of its potential activity (active complex). Only when DTNB treatments accounted for complex dissociation (>14 thiols oxidized per RI moiety) the released RI molecules exhibited the all-or-none oxidation behavior. By both kinetic and circular dichroism analyses, conformational changes have been evidenced for the transition from the inactive to the active form of RI-RNase complex. Relaxation of RI-RNase binding without major alterations in RI structure is proposed as responsible for complex activation. The results are discussed in terms of a model for the reversible regulation of RNase activity mediated by the redox status of RI.

Alkaline ribonuclease from the insect Ceratitis capitata

Abstract An alkaline ribonuclease has been purified from Ceratitis capitata larvae. The enzyme preparation gives a single band on SDS-polyacrylamide gel electrophoresis using both silver-stain and ribonucleolytic activity detection. This enzyme has been characterized as a cyclizing endonuclease with optimum pH value at 8.0–8.3. The enzyme preparation does not exhibit phosphatase activity. Poly(C) is the only homopolyribonucleotide degraded under standard assay conditions. Neither native nor denatured DNA is hydrolyzed by this enzyme. The protein is a single polypeptide chain of about 18 000 molecular weight; its secondary structure is composed of 19% α-helix, 10% β-structure, 71% aperiodic conformation with an average number of residues per helical segment of 9. The amino acid composition of this alkaline RNAase is also reported. Latent alkaline ribonuclease has been observed in crude insect homogenates. The insect enzyme is inhibited by the RNAase inhibitor from human placenta.

Selective silver staining of urease activity in polyacrylamide gels

A selective method for staining urease activity bands in nondenaturing polyacrylamide gels is described. It is based on the deposition of silver at the urease bands after incubation of gels in the presence of urea and photographic developers. Its highly sensitivity (up to 0.015 enzyme units, corresponding to 5 ng of purified urease) is based on both the silver deposition enhancement methodology and the developers used. The selectivity of the procedure is based on the local pH increase catalytically produced by the enzyme in the presence of urea. The densitometric scan of the enzyme bands gives a linear response at least in the range 0.015-0.300 urease units. This selective staining method is about 2.5 times more sensitive than the standard silver staining of proteins, in terms of detectable urease amount.

Ribonuclease-RNAase inhibitor complex from rat testis.: Purification of the RNAase inhibitor

The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated Mr value is 48,000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ.

Study of the RNA-degradating activities during the development of the insect Ceratitis capitata

1. Three different RNA-degrading activities have been characterized in the insect C. capitata. Two of them are non dependent on divalent cations and show acid and alkaline optimum pH values respectively. For the third one, the maximum activity is observed at pH 8.5, being this enzyme inhibited by EDTA. 2. Distribution of the enzyme levels during the development of the insect is reported. Results are interpreted in terms of the functional role of these enzymes.

Polyadenylated-RNA during the development of Ceratitis capitata

Abstract Polyadenylated-RNA has been isolated at different stages of the life cycle of the insect Ceratitis capitata using chromatography on oligo-dT-cellulose. The molecular characterization of this RNA fraction has been carried out by melting, sedimentation, electrophoretic and CD studies. The distribution of this polynucleotide fraction during the development has been related to physiological changes of this holometabolous insect. The results obtained are discussed in terms of the presence of RNA-degrading activities previously reported for this insect.

Urine metabolomics insight into acute kidney injury point to oxidative stress disruptions in energy generation and H 2 S availability

Acute kidney injury (AKI) is one of the main complications in acute care medicine and a risk factor for chronic kidney disease (CKD). AKI incidence has increased; however, its diagnosis has limitations and physiopathological mechanisms are underexplored. We investigated urine samples, aiming to identify major metabolite changes during human AKI evolution. Metabolic signatures found were further explored for a potential link to severity of injury. Twenty-four control subjects and 38 hospitalized patients with AKI were recruited and urine samples were collected at the time of diagnosis, during follow-up and at discharge. Nuclear magnetic resonance (NMR) was used in a first discovery phase for identifying potential metabolic differences. Target metabolites of interest were confirmed by liquid chromatography-mass spectrometry (LC-MS/MS) in an independent group. Underlying metabolic defects were further explored by kidney transcriptomics of murine toxic AKI. Urinary 2-hydroxybutyric acid, pantothenic acid, and hippuric acid were significantly downregulated and urinary N-acetylneuraminic acid, phosphoethanolamine, and serine were upregulated during AKI. Hippuric acid, phosphoethanolamine, and serine showed further downregulation/upregulation depending on the metabolite in acute tubular necrosis (ATN) AKI compared to prerenal AKI. Kidney transcriptomics disclosed decreased expression of cystathionase, cystathionine-β-synthase, and ethanolamine-phosphate cytidylyltransferase, and increased N-acetylneuraminate synthase as the potentially underlying cause of changes in urinary metabolites. A urinary metabolite panel identified AKI patients and provided insight into intrarenal events. A urine fingerprint made up of six metabolites may be related to pathophysiological changes in oxidative stress, energy generation, and H2S availability associated with AKI.Key messagesThe urinary metabolome reflects AKI evolution and severity of injury.Kidney transcriptomics revealed enzymatic expression changes.Enzymatic expression changes may be the potentially underlying cause of changes in urine metabolites.Identified metabolite changes link oxidative stress, energy generation, and H2S availability to AKI.

Characterization of the nucleolytic activity from the digestive juice of Ceratitis capitata larvae

1. 1. The ability of the digestive juice of Ceratitis capitata larvae to degrade polynucleotides has been studied: optimum pH, cationic dependence and kinetic studies of the hydrolytic reaction. 2. 2. The relative catalytic efficiency of the digestive juice against different polynucleotides is reported. 3. 3. The existence of analogous nucleolytic activities in other insects is discussed as a general feature in the diet digestion of these organisms.