A methodology for monitoring globular milk protein changes induced by ultrafiltration: a dual structural and functional approach.

Understanding filtration mechanisms at a molecular level is important for predicting structural and functional properties of globular milk proteins after membrane operations. This stage is thus highly decisive for the further development of membrane separations as an efficient alternative to chromatographic processes for the fractionation of milk proteins. In this study, we proposed an original and complete analytical package for the examination of the putative effect of filtration at both macroscopic and molecular levels. We then investigated the pertinence of this analytical package during ultrafiltration (UF) of globular milk proteins in both dead-end and crossflow modes. Reverse-phase HPLC combined with statistical computing was shown to be relevant for the assessment of even slight physically induced modifications. Adaptations of circular dichroism and solubility measurements, regarding their respective dependence on temperature and pH, were also useful for an accurate evaluation of functional modifications. At last, immunochemistry was proven to be a pertinent tool for the specific detection of modifications affecting a targeted protein, even in mixed solutions. Moreover, results obtained by such methods were shown to be coherent with data obtained from classical techniques such as fluorescence. For beta-lactoglobulin, some physically induced modifications were noticed in the permeate because of shear stress inside membrane pores. In the case of alpha-lactalbumin dead-end UF, permeation was shown to affect protein characteristics because of an increase in the relative calcium content responsible for a conformational transition from the apo-form to the holo-form of the protein. Finally, during crossflow UF with limited transmission of BSA, observations were coherent with a partial aggregation because of the circulation of proteins in the filtration pilot. Such a hypothesis corroborates results previously mentioned in the literature.

How microwaves can help to study membrane ageing.

This paper studies original protocols of rapid PES/PVP membrane NaOCl degradation allowing at reaching ageing states that are representative of industrial ageing. The long term objective is to propose basis for further fundamental studies aiming at the improvement of the impact of membrane ageing on behaviour in UF (fouling and cleaning mastering). The key of several protocols is the use of ageing acceleration thanks to microwave irradiation, either continuous or pulsed ones, that can be further associated (or not) with short ageing time in UF conditions. To evaluate the representativeness of obtained aged membranes, comparisons are achieved between pristine, voluntary laboratory aged membranes and an industrial membrane at the end of its service-life. Several physico-chemical analyses were used (ATR-FTIR, SEM-EDX, contact angle, SEC-HPLC). Evaluation of UF performances were made in UF of a model protein (Lysozyme, 14,300 g.mol-1). Finally, the proof of concept is done that conditions using MW exist to reach ageing state representative of industrial ageing.

On the impact of ethanol on the rejection and transfer mechanism during ultrafiltration of a charged macromolecule in water/ethanol.

Ultrafiltration (UF) is a sustainable membrane separation technique. It could be useful for the concentration/purification of bio-sourced molecules that are extracted either by pure ethanol or by water/ethanol mixtures. Nevertheless, the process optimization requires an in-depth understanding of the transfer mechanisms of solute through membranes, especially for charged solutes, that are nowadays not sufficiently documented. Previous studies achieved in aqueous media have shown that the rejection of charged solutes by an UF membrane involves at least three mechanisms: convection, diffusion and electrostatic interactions. The present study aims at a systematic analysis of the transfer mechanisms of a model protein (lysozyme) in water/ethanol mixtures (100/0-70/30 v/v) during UF by a zirconia inorganic membrane. The influence of the pH varying in the 4-9 range and of the ionic strength (I) is also discussed. The ionic strength I can be adjusted by addition of an indifferent electrolyte (NaCl) only aiming at the screening of the electrostatic interactions or by addition of a selectively adsorbed electrolyte(KH2PO4) that is able to change the isoelectric pH of the protein and thus to modulate the electrostatic interactions in a different way when compared to NaCl. Of course, both salts have an impact on the protein rejection in UF. The results are analysed using the CDE model previously developed in our group to explain the behaviour of a single protein during UF in water and accounting for convection, diffusion and electrophoretic migration. The applicability of the CDE model in water/ethanol mixtures up to 70/30 v/v is finally shown.

The biochemistry of memory: a new and specific hypothesis.

Recent studies have uncovered a synaptic process with properties required for an intermediate step in memory storage. Calcium rapidly and irreversibly increases the number of receptors for glutamate (a probable neurotransmitter) in forebrain synaptic membranes by activating a proteinase (calpain) that degrades fodrin, a spectrin-like protein. This process provides a means through which physiological activity could produce long-lasting changes in synaptic chemistry and ultrastructure. Since the process is only poorly represented in the brain stem, it is hypothesized to be responsible for those forms of memory localized in the telencephalon.

Entorhinal cortex lesions induce a decreased calcium transport in hippocampal mitochondria.

Lesions to the entorhinal afferent of the hippocampus in rats caused marked changes in calcium transport into mitochondria. Pyruvate-supported calcium transport into mitochondria from the denervated hippocampus was decreased to a larger extent than succinate-supported transport, and adenosine triphosphate-supported transport was not significantly modified. Although cytochrome oxidase and succinate dehydrogenase activities were not significantly changed by entorhinal lesions, pyruvate flux through pyruvate dehydrogenase was significantly decreased, and this effect was correlated with changes in pyruvate-supported calcium transport. The active portion of pyruvate dehydrogenase decreased, whereas total pyruvate dehydrogenase was not modified. These data suggest that denervation might initiate dendritic atrophy and subsequent growth responses by modifying calcium regulation through a change in the phosphorylation of pyruvate dehydrogenase.

Inhibitors of lysosomal enzymes: accumulation of lipofuscin-like dense bodies in the brain.

Injections of leupeptin (a thiol proteinase inhibitor) or chloroquine (a general lysosomal enzyme inhibitor) into the brains of young rats induced the formation of lysosome-associated granular aggregates (dense bodies) which closely resembled the ceroid-lipofuscin that accumulates in certain disease states and during aging. The dense material increased in a dose- and time-dependent fashion and was differentially distributed across brain regions and cell types. These observations provide clues to the origins of ceroid-lipofuscin and suggest means for studying the consequences of its accumulation.

Micromolar calcium stimulates proteolysis and glutamate binding in rat brain synaptic membranes.

Incubation of cortical synaptic membranes with low concentrations of calcium resulted in a decrease in the amount of a high-molecular-weight doublet protein and an increase in the sodium-independent binding of glutamate. Both effects were blocked by the thiol protease inhibitor leupeptin. These results suggest that calcium-induced proteolysis of membrane components regulates the number of glutamate receptors in neuronal membranes.

BDNF mRNA expression in the developing rat brain following kainic acid-induced seizure activity.

Brain-derived neurotrophic factor (BDNF) mRNA expression was studied in the hippocampus at various developmental stages in normal rats and following kainic acid (KA)-induced seizure activity. Systemic administration of KA strongly elevated BDNF mRNA levels in all hippocampal subregions after postnatal day 21. In contrast, even though KA induced intense behavioral seizure activity at postnatal day 8, the seizures were not associated with elevations of BDNF mRNA levels, indicating a clear dissociation between behavioral seizures and increases in BDNF mRNA levels and contradicting the view that BDNF mRNA expression is principally regulated by neuronal activity. In the dentate gyrus at postnatal day 13, intense BDNF mRNA expression was limited to a defined area at the border between granule cell and molecular layers, suggesting the possibility that segregation of BDNF mRNA into defined subcellular compartments may play a role in establishing the well-delineated patterns of innervation in the hippocampus.

Selective neuronal vulnerability in the hippocampus--a role for gene expression?

Proposed mechanisms of neurodegeneration focus generally on the triggering of toxic biochemical pathways by an increased intracellular concentration of Ca2+. Recent evidence also suggests that Ca2+ causes transcriptional activation of so-called 'cell-death genes'. Efforts to elucidate the basis of selective vulnerability have relied on animal models of delayed neuronal death in the hippocampus. Biochemical and morphological data indicate that delayed neuronal death is a form of programmed cell death, or apoptosis. Observations that specific genes are activated transcriptionally for prolonged times in neuronal populations that are undergoing delayed death suggest that active gene expression is part of the neuronal-death cascade. Although a direct causal role remains to be proven, evidence implicates certain genes in neuronal-death pathways.

Involvement of the secretory pathway for AMPA receptors in NMDA-induced potentiation in hippocampus.

A chemical form of synaptic potentiation was produced with a brief bath application of NMDA to rat hippocampal slices. Two methods were used to assess changes in membrane-bound AMPA receptors. Traditional subcellular fractionation was used to isolate synaptic membranes; alternatively, membrane receptors were cross-linked with the membrane-impermeable reagent bis(sulfosuccinimidyl) suberate, and levels of nonmembrane receptors were determined. In both cases, Western blots were used to determine the content of receptor subunits in various subcellular fractions. NMDA-induced potentiation was associated with increased levels of glutamate receptor 1 (GluR1) and GluR2/3 subunits of AMPA receptors in synaptic membrane preparations, whereas no change was observed in whole homogenates. Both KN-62, an inhibitor of calcium/calmodulin kinase, and calpain inhibitor III, a calpain inhibitor, inhibited NMDA-induced potentiation and changes in GluR1 and GluR2/3 subunits of AMPA receptors. Brefeldin A (BFA) inhibits protein trafficking between the Golgi apparatus and cell membranes. Pretreatment of hippocampal slices with BFA significantly decreased NMDA-induced potentiation and completely prevented an NMDA-induced increase in GluR1 levels in membrane fractions. Thus, the levels of GluR1 and GluR2/3 subunits of AMPA receptors are rapidly upregulated in synaptic membranes under conditions associated with potentiation of synaptic responses, and this upregulation requires a functional secretory pathway.

Endogenous phosphorylation and dephosphorylation of rat liver plasma membrane proteins, suggesting a 18 kDa phosphoprotein as a potential substrate for alkaline phosphatase.

Purified rat liver plasma membranes were incubated for 0-60 min with [gamma-32P]ATP and analysis of 32P-labeled proteins by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the presence of two shifted kinetic phenomena. The use of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinases, allowed the identification of one as the endogenous protein phosphorylation. The other was shown to be the labeling of two phospho-intermediate forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1.], which have apparent molecular masses of 151 and 135 kDa. Bromolevamisole, a potent inhibitor of the enzyme, stabilized these phospho-intermediates, and consequent on this inhibition the labelling of a 18 kDa phosphoprotein was augmented. So, when alkaline phosphatase was studied in its native plasma membrane environment, a specificity of this enzyme over the endogenous phosphoproteins was established.

The tight association of protein kinase CK2 with plasma membranes is mediated by a specific domain of its regulatory beta-subunit.

Previous immunocytochemical studies have shown that protein kinase CK2 is mostly detected both in the cytoplasm and the nucleus of most cells. In the present study, CK2 was detected in highly purified plasma membrane preparations from rat liver. The protein kinase could be released from the membranes by high salt extraction (>1 M NaCl). Plasma membranes prepared from SF9 insect cells expressing the alpha- and beta-subunits of CK2 also contained a significant amount of oligomeric CK2. Furthermore, it was demonstrated in this cell system as well as in rat liver plasma membranes, that the beta-subunit of the kinase is the targeting subunit which mediates the tight association of the enzyme to plasma membrane components. Binding studies using membranes and recombinant proteins corresponding to different regions of the beta-subunit suggest that a functional domain previously shown to be involved in the binding of polyamines may also participate to the binding of CK2 to membranes. Modification of membranes by trypsin and phospholipases indicated that the binding process may require both membrane protein(s) and phospholipids. Interestingly, it was observed that the amount of membrane-bound CK2 in liver of embryos and new born rats increases dramatically after birth and persists during the postnatal stages of development.

p53 accumulation due to down-regulation of ubiquitin: relevance for neuronal apoptosis.

The p53 tumor suppressor protein is a major regulator of cell growth arrest and apoptosis in response to DNA damage. Both p53 function and stability are tightly controlled by Mdm2, which binds to the p53 N-terminus and targets p53 for ubiquitin-mediated proteolysis. Previous studies suggest that adrenalectomy-induced neuronal apoptosis is p53-dependent. Here we demonstrate both nuclear accumulation and functional activation of p53 protein in apoptotic hippocampal neurons from adrenalectomized rats. Increased p53 expression occurred despite the accumulation of its negative regulator, Mdm2, and the formation of p53-Mdm2 complexes. The persistence of p53 expression was explained by a striking decrease in free ubiquitin in p53-positive neurons. The addition of exogenous ubiquitin to p53-Mdm2 complexes from apoptotic neurons restored p53 degradation. These findings demonstrate a novel mechanism of p53 stabilization mediated by decreased ubiquitin levels. Regulation of free ubiquitin may therefore be an effective way to modulate p53-dependent apoptosis in certain cell types.

Triggers and substrates of hippocampal synaptic plasticity.

It is widely assumed that behavioral learning reflects adaptive properties of the neuronal networks underlying behavior. Adaptive properties of networks in turn arise from the existence of biochemical mechanisms that regulate the efficacy of synaptic transmission. Considerable progress has been made in the elucidation of the mechanisms involved in synaptic plasticity at central synapses and especially those responsible for the phenomenon of long-term potentiation (LTP) of synaptic transmission in hippocampus. While the nature and the timing requirements of the triggering steps are reasonably well known, there is still a lot of uncertainty concerning the mechanisms responsible for the long-term changes. Several biochemical processes have been proposed to play critical roles in promoting long-lasting modifications of synaptic efficacy. This review examines first the triggers that are necessary to produce LTP in the hippocampus and then the different biochemical processes that have been considered to participate in the maintenance of LTP. Finally, we examine the relationships between LTP and behavioral learning.

Calcium dependent aspects of synaptic plasticity, excitatory amino acid neurotransmission, brain aging and schizophrenia: a unifying hypothesis.

(1) The functional and structural reorganization of dendritic spines by calcium activated proteases is postulated to play a causal role in the production of the phenomenology of brain aging and in particular in the development of pathology and degeneration. Excitatory neurotransmission appears to be essential for the development of irreversible synaptic changes. (2) One of the genes modified in schizophrenia is postulated to be directly or indirectly linked to the control of excitatory neurotransmission; possibly the normal switching on of the expression of the adult form of the NMDA receptor is altered, resulting in an inappropriate functioning of this receptor. This genetic characteristic might explain the apparent resistance of schizophrenic brains to aging.

Low levels of calpain activity in Chiroptera brain: implications for mechanisms of aging.

Calcium-dependent neutral proteases ("calpains") have been implicated in degenerative processes in muscles and neurons, suggesting that they might also play a role in age-related brain pathologies and perhaps in brain aging itself. Because Chiroptera exhibit an unusual maximum life span, relative to other mammalian orders, we investigated the activity of these enzymes in the brain of two species of bats. As in other mammals, brain calpain degrades many proteins associated with the cell cytoskeleton. However, enzyme activity is 5-7 fold lower in bat's brain than in a similar-sized mammal, such as the mouse. Moreover, the maximal life span of bats predicted from the equation relating calpain activity and maximal life span across a wide range of mammals is close to the observed values. These results strengthen the hypothesis that calpain activity is somehow linked to the rate at which brains age.

Oxygen free radicals in rat limbic structures after kainate-induced seizures.

Several indices of free radical generation were determined in limbic structures after kainate (KA)-induced seizure activity in adult and postnatal day (PND) 12 and 17 rats. Superoxide dismutase, catalase, and glutathione peroxidase activities were measured in piriform cortex and hippocampal subfields at 8, 16, 48 h, and 5 days after KA injection in adults and pups, and also at 3 weeks postinjection in adults. KA-induced seizure activity had no significant effect on enzyme activities in PND 12 and 17 rats. In adults, superoxide dismutase and catalase activities were significantly increased at 5 days after KA administration, and returned to preinjection levels by 3 weeks. Glutathione peroxidase activity was also increased significantly at 5 days postinjection, but remained elevated at 3 weeks. Lipid peroxidation, as indicated by malondialdehyde (MDA) concentration, exhibited an early significant increase at 8 and 16 h, followed at 48 h and 5 days by a significant decrease. At 3 weeks postinjection, MDA levels were still significantly decreased in CA3 and dentate gyrus. KA administration in PND 12 and 17 rats had no significant effect on MDA content. KA-induced seizure activity in adults also resulted in a large and sustained increase in protein oxidation in piriform cortex and hippocampus. The early increase in MDA and protein oxidation in adult rats strongly suggests the involvement of oxygen free radicals in the initial phases of KA-induced pathology, whereas the changes in scavenging enzyme activities and MDA content at 5 days and 3 weeks post KA injection possibly reflect glial proliferation subsequent to neuronal death.

Effect of Temperature and Calcium on the Binding Properties of the AMPA Receptor in Frozen Rat Brain Sections.

The elucidation of the mechanisms regulating the properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors is important for understanding glutamatergic transmission. Here we report that qualitative as well as quantitative analysis of tritiated ligand binding to the AMPA receptor on thin frozen rat brain tissue sections reveals the existence of several mechanisms regulating the binding properties of AMPA receptors. Preincubation of tissue sections at 35 degrees C results in a decreased amount of [3H]AMPA binding as compared to that measured following preincubation at 0 degrees C. The decrease in binding appears to be mainly localized to cell bodies as evaluated by autoradiography, and could be due to proteolysis. Preincubation with calcium at 35 degrees C produces increased levels of [3H]AMPA binding. The effect of calcium is mimicked by manganese and to a lesser extent by magnesium; it is concentration-dependent with a 50% effective concentration for calcium of approximately 150 microM, time-dependent and temperature-dependent. The calcium-induced increase in [3H]AMPA binding is different among various brain structures, being larger in area CA1 of the hippocampus and in the superficial layers of the cerebral cortex. The effect of calcium is partly reduced by preincubation with the calpain inhibitor leupeptin and potentiated by preincubation with purified calpain II. The calcium-induced increase in [3H]AMPA binding is associated with a decrease in the binding of an antagonist of AMPA receptors, [3H]6-nitro-7-cyanoquinoxaline-2,3-dione. The results indicate that the binding properties of the AMPA receptor are rapidly regulated by calcium-dependent processes, and possibly by calcium-dependent proteases. They suggest that modulation of the binding properties involves changes in the configuration of the receptor, producing opposite changes in the affinities of the receptor for agonists and antagonists. Finally, these results strengthen the hypothesis that changes in the properties of AMPA receptors might underlie various forms of synaptic plasticity.

Distribution of calcium-activated protease calpain in the rat brain.

Calpain is a calcium-activated neutral protease that degrades a number of cytoskeletal proteins. It may participate in the maintenance of the cytoskeleton and in the rapid turnover of structural proteins associated with synaptic plasticity. Calpain may also be involved in the neurodegeneration that accompanies aging and age-related diseases. To aid in the interpretation of disease-related alterations in staining patterns, the present study examined calpain's normal distribution in the mammalian brain and spinal cord. A monoclonal antibody was employed with the avidin-biotin-peroxidase immunocytochemical technique on samples of rat tissue. Glia (astrocytes, microglia) and virtually all neurons were immunopositive, although neuronal processes exhibited varying staining patterns. The axonal staining pattern depended upon either the origin or destination of the process: those axons remaining within the brain (e.g., corpus callosum) were only lightly immunoreactive, whereas spinal cord and peripheral axons (trigeminal nerve) were more darkly labeled. The architecture of the dendritic tree determined the dendritic staining pattern: neurons with prominent apical and basal dendritic trees (e.g., pyramidal cells) were immunolabeled along their entire extent; labeling of multipolar cells (e.g., hilar cells of dentate gyrus) was limited to the proximal dendrites. The ubiquitous distribution of calpain argues against a primary role for the enzyme in the regional pattern of neuronal death seen in Alzheimer's disease. An alteration in the concentration, localization, or inhibition of the enzyme could, however, lead to the abnormal accumulations of cytoskeletal elements seen with the disease.