Nuclear distribution in Alternaria tenuis

Nuclear distribution and behaviour during vegetative growth and spore formation inAlternaria tenuis was studied utilising the HCl — Giemsa staining technique. The vegetative mycelium and conidia are predominantly monokaryotic. Anastomoses, followed by nuclear migrations, have been recorded. Intercellular nuclear migrations have only been observed in germinating conidia. Nuclear behaviour during conidial formation indicates that the conidia are homokaryotic. Cytological differences have been found in different monocoidal isolates of A. tenuis.     

The complex regulation of human glud1 and glud2 glutamate dehydrogenases and its implications in nerve tissue biology

Mammalian glutamate dehydrogenase (GDH) is a housekeeping mitochondrial enzyme (hGDH1 in the human) that catalyses the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, thus interconnecting amino acid and carbohydrate metabolism. It displays an energy sensing mechanism, which permits enzyme activation under low cellular energy states. As GDH is at the crossroads of important metabolic pathways, a tight control of its activity is essential. Indeed, to fulfill its role in metabolism and cellular energetics, mammalian GDH has evolved into a highly regulated enzyme subject to allosteric modulation by diverse compounds. The recent emergence (<23million years ago) in apes and humans of a hGDH2 isoenzyme with distinct regulatory properties, as well as, the detection of gain-of-function variants in hGDH1 and hGDH2 that affect the nervous system, have introduced additional complexities. The properties of the two highly homologous human GDHs were studied using purified recombinant hGDH1 and hGDH2 obtained by expression of the corresponding cDNAs in Sf21 cells. Results showed that, in contrast to hGDH1 that maintains substantial basal activity (35-40% of its maximal capacity), hGDH2 displays low basal activity (3-8% of maximal) that is remarkably responsive to activation by rising levels of ADP and/or l-leucine. This is primarily due to the Arg443Ser evolutionary change, which also made hGDH2 markedly sensitive to estrogens and neuroleptic drugs. In contrast to hGDH1, which is subject to potent GTP inhibition, hGDH2 has dissociated its function from this energy switch, being able to metabolize glutamate even when the Krebs cycle generates GTP levels sufficient to inactivate the housekeeping hGDH1. Our data also show that spermidine, a polyamine thought to reduce oxidative stress and to prolong survival, and EGCG, a green tea polyphenol, inhibit hGDH2 at lower concentrations than hGDH1. The implications of these findings in nerve tissue biology are discussed.     

The human GLUD2 glutamate dehydrogenase and its regulation in health and disease

Whereas glutamate dehydrogenase in most mammals (hGDH1 in the human) is encoded by a single functional GLUD1 gene expressed widely, humans and other primates have acquired through retroposition an X-linked GLUD2 gene that encodes a highly homologous isoenzyme (hGDH2) expressed in testis and brain. Using an antibody specific for hGDH2, we showed that hGDH2 is expressed in testicular Sertoli cells and in cerebral cortical astrocytes. Although hGDH1 and hGDH2 have similar catalytic properties, they differ markedly in their regulatory profile. While hGDH1 is potently inhibited by GTP and may be controlled by the need of the cell for ATP, hGDH2 has dissociated its function from GTP and may metabolize glutamate even when the Krebs cycle generates GTP amounts sufficient to inactivate hGDH1. As astrocytes are known to provide neurons with lactate that largely derives from the Krebs cycle via conversion of glutamate to α-ketoglutarate, the selective expression of hGDH2 may facilitate metabolic recycling processes essential for glutamatergic transmission. As there is evidence for deregulation of glutamate metabolism in degenerative neurologic disorders, we sequenced GLUD1 and GLUD2 genes in neurologic patients and found that a rare T1492G variation in GLUD2 that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 interacted significantly with Parkinson's disease (PD) onset. Thus, in two independent Greek and one North American PD cohorts, Ser445Ala hemizygous males, but not heterozygous females, developed PD 6-13 years earlier than subjects with other genotypes. The Ala445-hGDH2 variant shows enhanced catalytic activity that is resistant to modulation by GTP, but sensitive to inhibition by estrogens. These observations are thought to suggest that enhanced glutamate oxidation by the Ala445-hGDH2 variant accelerates nigral cell degeneration in hemizygous males and that inhibition of the overactive enzyme by estrogens protects heterozygous females. We then evaluated the interaction of estrogens and neuroleptic agents (haloperidol and perphenazine) with the wild-type hGDH1 and hGDH2 and found that both inhibited hGDH2 more potently than hGDH1 and that the evolutionary Arg443Ser substitution was largely responsible for this sensitivity. Hence, the properties acquired by hGDH2 during its evolution have made the enzyme a selective target for neuroactive steroids and drugs, providing new means for therapeutic interventions in disorders linked to deregulation of this enzyme.     

Heterogeneous Cellular Distribution of Glutamate Dehydrogenase in Brain and in Non-neural Tissues

Mammalian glutamate dehydrogenase (GDH) is an evolutionarily conserved enzyme central to the metabolism of glutamate, the main excitatory transmitter in mammalian CNS. Its activity is allosterically regulated and thought to be controlled by the need of the cell for ATP. While in most mammals, GDH is encoded by a single GLUD1 gene that is widely expressed (housekeeping; hGDH1 in the human), humans and other primates have acquired via retroposition a GLUD2 gene encoding an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. Whereas hGDH1 shows high levels of expression in the liver, hGDH2 is expressed in human testis, brain and kidney. Recent studies have provided significant insight into the functional adaptation of hGDH2. This includes resistance to GTP control, enhanced sensitivity to inhibition by estrogens and other endogenous allosteric effectors, and ability to function in a relatively acidic environment. While inhibition of hGDH1 by GTP, derived from Krebs cycle, represents the main mechanism by which the flux of glutamate through this pathway is regulated, dissociation of hGDH2 from GTP control may provide a biological advantage by permitting enzyme function independently of this energy switch. Also, the relatively low optimal pH for hGDH2 is suited for transmitter glutamate metabolism, as glutamate uptake by astrocytes leads to significant mitochondrial acidification. Although mammalian GDH is a housekeeping enzyme, its levels of expression vary markedly among the various tissues and among the different types of cells that constitute the same organ. In this paper, we will review existing evidence on the cellular and subcellular distribution of GDH in neural and non-neural tissues of experimental animals and humans, and consider the implications of these findings in biology of these tissues. Special attention is given to accumulating evidence that glutamate flux through the GDH pathway is linked to cell signaling mechanisms that may be tissue-specific.     

Study of structure-function relationships in human glutamate dehydrogenases reveals novel molecular mechanisms for the regulation of the nerve tissue-specific (GLUD2) isoenzyme

In mammalian brain, glutamate dehydrogenase (GDH) is located predominantly in astrocytes, where is thought to play a role in transmitter glutamate's metabolism. Human GDH exists in GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) isoforms, which share all but 15 out of their 505 amino acids. The GLUD1 GDH is potently inhibited by GTP, whereas the GLUD2 enzyme is resistant to this compound. On the other hand, the GLUD2 isoform assumes in the absence of GTP a conformational state associated with little catalytic activity, but it remains amenable to full activation by ADP and/or L-leucine. Site-directed mutagenesis of the GLUD1 gene at sites that differ from the corresponding residues of the GLUD2 gene showed that replacement of Gly456 by Ala made the enzyme resistant to GTP (IC(50)=2.8+/-0.15 microM) compared to the wild-type GDH (IC(50)=0.19+/-0.01 microM). In addition, substitution of Ser for Arg443 virtually abolished basal activity and rendered the enzyme dependent on ADP for its function. These properties may permit the neural enzyme to be recruited under conditions of low energy charge (high ADP:ATP ratio), similar to those that prevail in synaptic astrocytes during intense glutamatergic transmission. Hence, substitution of Ser for Arg443 and Ala for Gly456 are the main evolutionary changes that led to the adaptation of the GLUD2 GDH to the unique metabolic needs of the nerve tissue.     

Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC₅₀ = 0.1–0.3 μm) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC₅₀ = 0.08 ± 0.01 μm) and with ∼18-fold lower affinity hGDH1 (IC₅₀ = 1.67 ± 0.06 μm; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC₅₀ = 1.53 ± 0.24 μm) than for hGDH1 (IC₅₀ = 26.94 ± 1.07 μm; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.     

Substitution of Ser for Arg-443 in the regulatory domain of human housekeeping (GLUD1) glutamate dehydrogenase virtually abolishes basal activity and markedly alters the activation of the enzyme by ADP and L-leucine

Human glutamate dehydrogenase (GDH) exists in GLUD1 (housekeeping) and in GLUD2-specified (brain-specific) isoforms, which differ markedly in their basal activity and allosteric regulation. To determine the structural basis of these functional differences, we mutagenized the GLUD1 GDH at four residues that differ from those of the GLUD2 isoenzyme. Functional analyses revealed that substitution of Ser for Arg-443 (but not substitution of Thr for Ser-331, Leu for Met-370, or Leu for Met-415) virtually abolished basal activity and totally abrogated the activation of the enzyme by l-leucine (1-10 mm) in the absence of other effectors. However, when ADP (0.025-0.1 mm) was present in the reaction mixture, l-leucine (0.3-6.0 mm) activated the mutant enzyme up to >2,000%. The R443S mutant was much less sensitive to ADP (SC(50) = 383.9 +/- 14.6 microm) than the GLUD1 GDH (SC(50) = 31.7 +/- 4.2 microm; p < 0.001); however, at 1 mm ADP the V(max) for the mutant (136.67 micromol min(-1) mg(-1)) was comparable with that of the GLUD1 GDH (152.95 micromol min(-1) mg(-1)). Varying the composition and the pH of the reaction buffer differentially affected the mutant and the wild-type GDH. Arg-443 lies in the "antenna" structure, in a helix that undergoes major conformational changes during catalysis and is involved in intersubunit communication. Its replacement by Ser is sufficient to impair both the catalytic and the allosteric function of human GDH.