Structural studies of hydroxycitrates and their relevance to certain enzymatic mechanisms.

The crystal structures of the ethylenediamine salts of two diastereoisomeric hydroxycitratesy are described, and their conformations in the solid state are analyzed. In both structures, the HOCCOH torsion angle is approximately 60 ° as found for many tartrates and mesotartrates. The presence of three carboxyl groups and two hydroxyl groups in hydroxycitrates leads to 10 possible types of tridentate metal chelates. Since bacterial citrate lyase and ATP citrate lyase require metal ions, the possible geometries of hydroxycitrate chelation have been compared with those of citrate, and as a result, some information on the geometry of each enzymic active site has been obtained. If the hydroxycitrate binds in the same manner as citrate, the C(3)&;z.sbnd;C(4) bond will be in the correct position to be cleaved. Other modes of binding of hydroxycitrate, if they can be accommodated in the active site of the enzyme, are nonproductive and compete with the citrate-like mode causing inhibition. It is possible, in these alternate modes of binding of hydroxycitrates, for additional binding to side chains in the active site of the enzyme to occur, resulting in extremely potent inhibition.     

Cellular mechanisms of brain hypoglycemia

Data on intracellular processes induced by a low glucose level in nerve tissue are presented. The involvement of glutamate and adenosine receptors, mitochondria, reactive oxygen species (ROS), and calcium ions in the development of hypoglycemia-induced damage of neurons is considered. Hypoglycemia-induced calcium overload of neuronal mitochondria is suggested to be responsible for the increased ROS production by mitochondria.     

Cellular energy metabolism during fetal development. VI. Fatty acid oxidation by developing brain.

We have investigated fatty acid oxidation and development profiles of palmitoyl-CoA synthetase and carnitine palmitoyltransferase in homogenates of developing rat brain. Palmitate showed a peak rate of oxidation between 10 days and the time of weaning, after which activity declined to adult levels. Acetate oxidation increased until Day 10, plateaued until Day 18 when it increased sharply and remained elevated through Day 25 before declining to the adult level. Leucine oxidation also showed a late peak as compared with palmitate. Palmitoyl-CoA synthetase activity was highest in late fetal development and in the newborn after which activity declined gradually to adult levels. Carnitine palmitoyltransferase activity peaked at 10–15 days of age similar to the profile obtained for long chain fatty acid oxidation. During the period of peak fatty acid oxidation, cytochrome oxidase activity increased twofold but the developmental increase in fatty acid oxidation and enzyme levels was much greater than the increase in mitochondrial number. These data suggest that during periods of high fat intake in the suckling rat the brain has an increased capacity for long chain fatty acid oxidation and that in addition to ketone bodies and leucine, fatty acids may be utilized as an alternative substrate in developing brain.     

Cellular mechanisms of vitamin E uptake: relevance in alpha-tocopherol metabolism and potential implications for disease

alpha-Tocopherol is an essential micronutrient involved in various oxidative stress-related processes. Because of its hydrophobic nature, alpha-tocopherol is transported in plasma lipoproteins, and the pathways involved in its cellular uptake are closely related to the lipoprotein metabolism. alpha-Tocopherol transfer from plasma to cells can occur by different mechanisms such as uptake facilitated by lipid transfer proteins and lipases, receptor-mediated lipoprotein endocytosis, and selective lipid uptake. Here we discuss recent progress in understanding the physiological and pathophysiological relevance of these different pathways for cellular uptake of vitamin E in vivo. This review is mainly focused on the role of the scavenger receptor class B type I (SR-BI) on alpha-tocopherol metabolism and its potential implications for disease conditions.     

Cellular mechanisms of estradiol-mediated masculinization of the brain

The sexual differentiation of reproductive physiology and behavior in the rodent brain is largely determined by estradiol aromatized from testicular androgens. The cellular mechanisms by which estradiol masculinizes the brain are beginning to emerge and revealing novel features of brain development that are highly region-specific. In the preoptic area, the major site controlling male sexual behavior, estradiol increases the level of the COX-2 enzyme and its product, prostaglandin E2 which promotes dendritic spine synaptogenesis. In the ventromedial nucleus of the hypothalamus, the major site controlling female reproductive behavior, estradiol promotes glutamate release from synaptic terminals, activating NMDA receptors and the MAP kinase pathway. In the arcuate nucleus, a major regulator of anterior pituitary function, estradiol increases GABA synthesis, altering the morphology of neighboring astrocytes and reducing formation of dendritic spines synapses. Glutamate, GABA and the importance of neuronal-astrocytic cross-talk are emerging as common aspects of masculinization. Advances are also being made in the mechanistic basis of female brain development, although the challenges are far greater.     

Cellular and tissue distribution of potassium: Physiological relevance,mechanisms and regulation

Potassium (K⁺) is the most important cationic nutrient for all living organisms. Its cellular levels are significant (typically around 100 mM) and are highly regulated. In plants K⁺ affects multiple aspects such as growth, tolerance to biotic and abiotic stress and movement of plant organs. These processes occur at the cell, organ and whole plant level and not surprisingly, plants have evolved sophisticated mechanisms for the uptake, efflux and distribution of K⁺ both within cells and between organs.     

Cellular compartments of GABA in brain and their relationship to anticonvulsant activity

Summary The effects of GABA-elevating agents were examined with respect to the cellular compartments in which GABA increases occurred and the brain region(s) that mediate the anticonvulsant activity of these compounds. Changes in GABA occurring in the presence and absence of GABAergic nerve terminals were estimated in vivo using rats in which the GABA projection to the substantia nigra (SN) was destroyed on one side of the brain. One week post-operatively, the GABA concentration in the denervated SN was 10–20% of control. The net increase in GABA content of the denervated SN was compared to that of the intact SN after intraperitoneal injection of amino-oxacetic acid (AOAA), di-n-propylacetate (DPA) and -vinyl GABA (GVG). In the intact SN, all drugs produced significant increases in GABA. In the denervated SN, both AOAA and GVG produced marked increases in GABA (nearly equivalent to those obtained in the intact SN) whereas DPA was without effect. It therefore appears that the DPA-induced elevation of GABA depends upon the presence of GABAergic nerve terminals whereas AOAA and GVG primarily elevate GABA in non-nerve terminal compartments. An increase in GABA associated with nerve terminals was obtained with GVG only after a latency of more than 12 h following a single injection. The time course of elevation of nerve terminal-associated GABA coincided with the time course of anticonvulsant action of GVG; both effects were maximal at 60 h after a single injection. Taken together, our results indicate that the ability of DPA, AOAA and GVG to protect against chemically- and electrically-induced seizures is directly correlated with increases in nerve terminal GABA and not related to increases in other GABA compartments.Localization of the anatomical site that mediates anticonvulsant activity was examined using intracerebral injections of GVG into fore-, mid-and hindbrain areas. Blockade of tonic hindlimb extension in the maximal electroshock test and blockade of tonic and clonic seizures produced by pentylenetetrazol and bicuculline was obtained by microinjection of GVG (10 µg) into the ventral tegmental area of the midbrain. Injections of GVG (10–40 µg) into forebrain areas (striatum, thalamus) or into hindbrain (pontine tegmentum) were without anticonvulsant activity. Anticonvulsant effects of midbrain GVG were correlated with GABA elevation (3–4 fold) within a 1.5 mm radius of the injection site; these effects were obtained within 6 h and lasted three to four days after a single treatment. After four days seizure activity returned to control. No changes in spontaneous motor activity or reflexes accompanied the GVG injections. Similar but shorter lasting anticonvulsant effects were obtained with the direct GABA receptor agonist muscimol (50 ng) injected into the midbrain site. On the other hand, doses of muscimol up to 500 ng placed in the rostral pontine tegmentum were without anticonvulsant effect, despite the appearance of marked sedation.The time to peak anticonvulsant activity after midbrain microinjection of GVG (6 h) was considerably more rapid than that after intraperitoneal injection (60 h). Compartmental analysis revealed that nerve terminal associated GABA was elevated by 6 h after GVG when the direct microinjection route was used. These results suggest that GABAergic synapses in the midbrain may be critically involved in the control of seizure propagation.     

Amyloid-fibril formation. Proposed mechanisms and relevance to conformational disease.

The phenomenon of the transformation of proteins into amyloid-fibrils is of interest, firstly, because it is closely connected to the so-called conformational diseases, many of which are hitherto incurable, and secondly, because it remains to be explained in physical terms (energetically and structurally). The process leads to fibrous aggregates in the form of extracellular amyloid plaques, neuro-fibrillary tangles and other intracytoplasmic or intranuclear inclusions. In this review, basic principles common to the field of amyloid fibril formation and conformational disease are underlined. Existing models for the mechanism need to be tested by experiment. The kinetic and energetic bases of the process are reviewed. The main controversial issue remains the coexistence of more than one protein conformation. The possible role of oligomeric intermediates, and of domain-swapping is also discussed. Mechanisms for cellular defence and novel therapies are considered.     

Proteomic analysis and functional characterization of mouse brain mitochondria during aging reveal alterations in energy metabolism

Mitochondria are the main cellular source of reactive oxygen species and are recognized as key players in several age‐associated disorders and neurodegeneration. Their dysfunction has also been linked to cellular aging. Additionally, mechanisms leading to the preservation of mitochondrial function promote longevity. In this study we investigated the proteomic and functional alterations in brain mitochondria isolated from mature (5 months old), old (12 months old), and aged (24 months old) mice as determinants of normal “healthy” aging. Here the global changes concomitant with aging in the mitochondrial proteome of mouse brain analyzed by quantitative mass‐spectrometry based super‐SILAC identified differentially expressed proteins involved in several metabolic pathways including glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Despite these changes, the bioenergetic function of these mitochondria was preserved. Overall, this data indicates that proteomic changes during aging may compensate for functional defects aiding in preservation of mitochondrial function. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001370 ( http://proteomecentral.proteomexchange.org/dataset/PXD001370 ).     

ROS-mediated mechanisms of autophagy stimulation and their relevance in cancer therapy

Mounting evidence suggests that reactive oxygen species (ROS) are multifaceted signalling molecules implicated in a variety of cellular programs during physiological as well as pathological conditions. Recently, ROS produced endogenously, by deranged metabolism of cancer cells, or exogenously, by ROS-generating drugs, have been shown to promote macroautophagy, a lysosomal pathway of self-degradation with essential prosurvival functions. Several molecular aspects of the modulation of autophagy pathways by ROS have been revealed in the past years and it is now clear that these processes are mutually linked and play a crucial role in cancer progression and in response to cancer therapeutics. In this review we address the molecular mechanisms underlying the activation of autophagy pathways by ROS and focus on the role of autophagy in cancer cells responding to ROS-producing agents, which are utilized as a therapeutic modality to kill cancer cells.     

Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance.

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.     

Regional enzyme development in rat brain. Enzymes of energy metabolism.

The development of several key enzymes of pyruvate and 3-hydroxybutyrate metabolism and of the tricarboxylic acid cycle was studied in six regions (cerebellum, medulla oblongata and pons, hypothalamus, striatum, mid-brain and cortex) of the neonatal, suckling and adult rat brain (2 days before birth to 60 days after birth). The enzymes whose developmental patterns were studied were: pyruvate dehydrogenase (EC 1.2.4.1), 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and fumarase (EC 4.2.1.2). Citrate synthase, isocitrate dehydrogenase and pyruvate dehydrogenase develop as a cluster in each region, although the pyruvate dehydrogenase appears to lag slightly behind the others. As with the glycolytic-enzyme cluster [Leong & Clark (1984) Biochem. J. 218, 131-138] the timing of the development of the activity of this group of enzymes varies from region to region; 50% of the adult activity developed first in the medulla oblongata, followed by the hypothalamus, striatum and mid-brain, and then in the cortex and cerebellum respectively. The 3-hydroxybutyrate dehydrogenase activity also develops earlier in the medulla oblongata than in the other regions. The results are discussed with respect to the neurophylogenetic development of the brain regions studied and the importance of the development of the enzymes of aerobic glycolysis in relationship to the development of neurological maturation.     

Paradoxical sleep deprivation: effects on brain energy metabolism.

A study was made of brain nucleotides and glycolytic intermediates in paradoxical sleep (PS)-deprived and recovery-sleeping rats. It was observed that PS deprivation of 24 h produced a fall in glucose, glucose 6-phosphate and pyruvate in cerebral frontal lobes. After three hours of recovery sleep all values returned toward their predeprivational levels. In cerebellar hemispheres ATP was increased, while glucose 6-phosphate and pyruvate were decreased. After three hours of recovery sleep, glucose 6-phosphate was increased and pyruvate decreased, indicating restoration of glycogen and creatine phosphate respectively.     

Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity

Flavonoids have been proposed to act as beneficial agents in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. The biological effect of these polyphenols and their in vivo circulating metabolites will ultimately depend on the extent to which they associate with cells, either by interactions at the membrane or more importantly their uptake. This review summarises the current knowledge on the cellular uptake of flavonoids and their metabolites with particular relevance to further intracellular metabolism and the generation of potential new bioactive forms. Uptake and metabolism of the circulating forms of flavanols, flavonols, and flavanones into cells of the skin, the brain, and cancer cells is reviewed and potential biological relevance to intracellular formed metabolites is discussed.     

Human brain glycogen content and metabolism: implications on its role in brain energy metabolism

The adult brain relies on glucose for its energy needs and stores it in the form of glycogen, primarily in astrocytes. Animal and culture studies indicate that brain glycogen may support neuronal function when the glucose supply from the blood is inadequate and/or during neuronal activation. However, the concentration of glycogen and rates of its metabolism in the human brain are unknown. We used in vivo localized 13C-NMR spectroscopy to measure glycogen content and turnover in the human brain. Nine healthy volunteers received intravenous infusions of [1-(13)C]glucose for durations ranging from 6 to 50 h, and brain glycogen labeling and washout were measured in the occipital lobe for up to 84 h. The labeling kinetics suggest that turnover is the main mechanism of label incorporation into brain glycogen. Upon fitting a model of glycogen metabolism to the time courses of newly synthesized glycogen, human brain glycogen content was estimated at approximately 3.5 micromol/g, i.e., three- to fourfold higher than free glucose at euglycemia. Turnover of bulk brain glycogen occurred at a rate of 0.16 micromol.g-1.h-1, implying that complete turnover requires 3-5 days. Twenty minutes of visual stimulation (n=5) did not result in detectable glycogen utilization in the visual cortex, as judged from similar [13C]glycogen levels before and after stimulation. We conclude that the brain stores a substantial amount of glycogen relative to free glucose and metabolizes this store very slowly under normal physiology.