Irreversible thermoinactivation of ribonuclease‐A by soft‐hydrothermal processing

Ribonuclease (RNase), which often represents molecular biological contamination, is a thermostable enzyme. When RNase is heated at 121°C by autoclave sterilization for 20 min, it does not lose its activity. However, the nature of the molecular events by which the irreversible denaturation occurs remains unknown. The purpose of this study was to elucidate the molecular mechanisms of irreversible thermal denaturation of RNase A and to develop an advanced sterilization method using soft‐hydrothermal processing, which has the advantages of improved safety and cost‐efficiency. The enzymatic activity of RNase was measured using polyacrylamide gel electrophoresis with torula yeast RNA. We evaluated the temperature and time course of irreversible thermoinactivation of RNase by normal autoclaving, hot‐air sterilization, and soft‐hydrothermal processing that had been controlled to the desired steam saturation ratio. The results indicated that RNase A was deactivated by autoclave sterilization (121°C, 20 min) immediately after treatment, but was reactivated over time. Hot‐air sterilization (180°C, atmospheric pressure, 60 min) produced results similar to that of autoclave sterilization. In contrast, RNase A was irreversibly thermoinactivated by soft‐hydrothermal processing (110°C, 20 min) at 100% steam saturation ratio. We also determined that the mechanism of irreversible thermoinactivation of RNase A involved hydrolysis and deamidation under this condition at a steam saturation ratio of more than 100%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

PAPP‐A: a new anti‐aging target?

This article focuses on the role of PAPP‐A in mammalian aging. It introduces PAPP‐A and a little of its history, briefly discusses the function of PAPP‐A in the insulin‐like growth factor (IGF) system and the regulators of PAPP‐A expression, and then reviews data concerning PAPP‐A in aging and age‐related diseases especially in regard to the PAPP‐A knockout (KO) mouse. The PAPP‐A KO mouse is a valuable new model to test hypotheses concerning the control of the tissue availability of IGF, independent from systemic levels, on healthspan as well as lifespan.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Novel 3‐Oxo‐ and 3,24‐Dinor‐2,4‐secooleanane‐Type Triterpenes from Terminalia ivorensis A. Chev.

Two new oleanane‐type triterpenes named ivorengenin A (=3‐oxo‐2α,19α,24‐trihydroxyolean‐12‐en‐28‐oic acid; 1 ) and ivorengenin B (=4‐oxo‐19α‐hydroxy‐3,24‐dinor‐2,4‐secoolean‐12‐ene‐2,28‐dioic acid; 2 ), together with five known compounds, arjungenin, arjunic acid, betulinic acid, sericic acid, and oleanolic acid, were isolated from the barks of Terminalia ivorensis A. Chev . (Combretaceae). Their structures were established on the basis of 1D‐ and 2D‐NMR data, and mass spectrometry. A biogenetic pathway to the formation of these compounds from sericic acid, isolated as the major compound from this plant, was proposed. The antioxidant activities of different compounds were investigated by means of the 2,2‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assays, and IC₅₀ values were calculated and compared with Trolox activity. Antiproliferative activities of the isolated compounds were also evaluated against MDA‐MB‐231, PC3, HCT116, and T98G human cancer cell lines, against which the compounds showed significant cytotoxic activities.     

Association between high‐sensitivity C‐reactive protein,lipoprotein‐associated phospholipase A2 and carotid atherosclerosis: A cross‐sectional study

High‐sensitivity C‐reactive protein (hs‐CRP) and lipoprotein‐associated phospholipase A2 (Lp‐PLA2) have been reported to be independent predictors of atherosclerosis. However, whether the combination of these two markers can improve the prediction of atherosclerosis is unknown. This study aimed to evaluate the association between combining hs‐CRP and Lp‐PLA2 and predicting carotid atherosclerosis. A total of 1982 participants aged ≥40 years were included in this study. Hs‐CRP and Lp‐PLA2 were measured by a high‐sensitivity nephelometry assay and quantitative sandwich enzyme‐linked immunosorbent assay, respectively. Ultrasonography was performed on the bilateral carotid arteries to evaluate stenosis and plaques. Multivariable logistic regression models were used to analyse the association between the combination of the hs‐CRP and Lp‐PLA2 levels and carotid plaques and stenosis. A total of 1579 (79.7%) and 181 (9.1%) subjects had carotid plaques and carotid stenosis, respectively. The group with high hs‐CRP and Lp‐PLA2 levels had the highest prevalence of carotid plaques (90.6%) and stenosis (20.8%). A significant association was found between high hs‐CRP and Lp‐PLA2 levels and carotid stenosis (adjusted odds ratio [OR]: 2.39; 95% confidence interval [CI]: 1.13‐5.09), but this combination was not associated with carotid plaques (OR: 2.62, 95% CI: 0.93‐7.38). The results suggested that the combination of hs‐CRP and Lp‐PLA2 were better predictors than either protein alone with regard to carotid atherosclerosis.     

Fetuin‐A in the developing brain

The serum protein fetuin‐A is essential for mineral homeostasis and shows immunomodulatory functions, for example by binding to TGF superfamily proteins. It proved neuroprotective in a rat stroke model and reduced lethality after systemic lipopolysaccharide challenge in mice. Serum fetuin‐A concentrations are highest during intrauterine life. Different species show intrauterine cerebral fetuin‐A immunoreactivity, suggesting a contribution to brain development. We therefore aimed at specifying fetuin‐A immunoreactivity in brains of newborn rats (age P0–P28) and human neonates (20–40 weeks of gestation). In humans and rats, fetuin‐A was found in cortex, white matter, subplate, hippocampus, subventricular zone, and ependymal cells which supports a global role for brain function. In rats, overall fetuin‐A immunoreactivity decreased with age. At P0 fetuin‐A immunoreactivity affected most brain structures. Thereafter, it became increasingly restricted to distinct cells of the hippocampus, cingular gyrus, periventricular stem cell layer, and ependyma. In ependymal cells the staining pattern complied with active transependymal transport from cerebrospinal fluid. Double immunofluorescence studies revealed colocalization with NeuN (mature neurons), beta III tubulin (immature neurons), GFAP (astrocytes), and CD68 (activated microglia). This points to a role of fetuin‐A in different brain functional systems. In human neonatal autopsy cases, frequently affected from severe neurological and non‐neurological diseases, fetuin‐A immunoreactivity was heterogeneous and much less associated with age than in healthy tissues studied earlier, suggesting an impact of exogeneous noxious factors on fetuin‐A regulation. Further research on the role of fetuin‐A in the neonatal brain during physiological and pathological conditions is recommended. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 354–369, 2013     

Nicotinamide‐rich diet improves physical endurance by up‐regulating SUR2A in the heart

SUR2A is an ATP‐binding protein that serves as a regulatory subunit of cardioprotective ATP‐sensitive K⁺ (K_ATP) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled K_ATP channels, we have suggested that nicotinamide‐rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide‐rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild‐type mice on nicotinamide‐rich diet. The experiments focused on action membrane potential and intracellular Ca²⁺ concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal K_ATP channels and steady Ca²⁺ levels in cardiomyocytes in response to β‐adrenergic stimulation. In contrast, the same challenge in the wild‐type was characterized by a lack of the channel activation and rise in intracellular Ca²⁺. Nicotinamide‐rich diet was ineffective to increase physical endurance in mice lacking K_ATP channels. This study has shown that nicotinamide‐rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide‐rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.     

S100A10‐Mediated Translocation of Annexin‐A2 to SNARE Proteins in Adrenergic Chromaffin Cells Undergoing Exocytosis

In neuroendocrine cells, annexin‐A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)‐containing lipid microdomains that are required for calcium‐regulated exocytosis. As soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin‐A2‐induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin‐A2 to the plasma membrane, where it colocalizes with SNAP‐25 and S100A10. Cross‐linking experiments performed in stimulated chromaffin cells indicate that annexin‐A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle‐associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin‐A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin‐A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)‐bisphosphate (PIP₂) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin‐A2‐mediated lipid microdomains and SNAREs during exocytosis.     

A study of the possible association between adenosine A2A receptor gene polymorphisms and attention‐deficit hyperactivity disorder traits

The adenosine A_2A receptor (ADORA2A) is linked to the dopamine neurotransmitter system and is also implicated in the regulation of alertness, suggesting a potential association with attention‐deficit hyperactivity disorder (ADHD) traits. Furthermore, animal studies suggest that the ADORA2A may influence ADHD‐like behavior. For that reason, the ADORA2A gene emerges as a promising candidate for studying the etiology of ADHD traits. The aim of this study was to examine the relationship between ADORA2A gene polymorphisms and ADHD traits in a large population‐based sample. This study was based on the Child and Adolescent Twin Study in Sweden (CATSS), and included 1747 twins. Attention‐deficit hyperactivity disorder traits were assessed through parental reports, and samples of DNA were collected. Associations between six single nucleotide polymorphisms (SNPs) and ADHD traits were examined, and results suggested a nominal association between ADHD traits and three of these SNPs: rs3761422, rs5751876 and rs35320474. For one of the SNPs, rs35320474, results remained significant after correction for multiple comparisons. These results indicate the possibility that the ADORA2A gene may be involved in ADHD traits. However, more studies replicating the present results are warranted before this association can be confirmed .