Genetic variant rs17225178 in the ARNT2 gene is associated with Asperger Syndrome

Background Autism Spectrum Conditions (ASC) are neurodevelopmental conditions characterized by difficulties in communication and social interaction, alongside unusually repetitive behaviours and narrow interests. Asperger Syndrome (AS) is one subgroup of ASC and differs from classic autism in that in AS there is no language or general cognitive delay. Genetic, epigenetic and environmental factors are implicated in ASC and genes involved in neural connectivity and neurodevelopment are good candidates for studying the susceptibility to ASC. The aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) gene encodes a transcription factor involved in neurodevelopmental processes, neuronal connectivity and cellular responses to hypoxia. A mutation in this gene has been identified in individuals with ASC and single nucleotide polymorphisms (SNPs) have been nominally associated with AS and autistic traits in previous studies. Methods In this study, we tested 34 SNPs in ARNT2 for association with AS in 118 cases and 412 controls of Caucasian origin. P values were adjusted for multiple comparisons, and linkage disequilibrium (LD) among the SNPs analysed was calculated in our sample. Finally, SNP annotation allowed functional and structural analyses of the genetic variants in ARNT2. We tested the replicability of our result using the genome-wide association studies (GWAS) database of the Psychiatric Genomics Consortium (PGC). Results We report statistically significant association of rs17225178 with AS. This SNP modifies transcription factor binding sites and regions that regulate the chromatin state in neural cell lines. It is also included in a LD block in our sample, alongside other genetic variants that alter chromatin regulatory regions in neural cells. Conclusions These findings demonstrate that rs17225178 in the ARNT2 gene is associated with AS and support previous studies that pointed out an involvement of this gene in the predisposition to ASC.

Abamectin affects the bioenergetics of liver mitochondria: A potential mechanism of hepatotoxicity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of mitochondria and biotransformation in abamectin-induced cytotoxicity in isolated rat hepatocytes

Abamectin (ABA), which belongs to the family of avermectins, is used as a parasiticide; however, ABA poisoning can impair liver function. In a previous study using isolated rat liver mitochondria, we observed that ABA inhibited the activity of adenine nucleotide translocator and FoF1-ATPase. The aim of this study was to characterize the mechanism of ABA toxicity in isolated rat hepatocytes and to evaluate whether this effect is dependent on its metabolism. The toxicity of ABA was assessed by monitoring oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, cell viability, intracellular Ca2+ homeostasis, release of cytochrome c, caspase 3 activity and necrotic cell death. ABA reduces cellular respiration in cells energized with glutamate and malate or succinate. The hepatocytes that were previously incubated with proadifen, a cytochrome P450 inhibitor, are more sensitive to the compound as observed by a rapid decrease in the mitochondrial membrane potential accompanied by reductions in ATP concentration and cell viability and a disruption of intracellular Ca2+ homeostasis followed by necrosis. Our results indicate that ABA biotransformation reduces its toxicity, and its toxic action is related to the inhibition of mitochondrial activity, which leads to decreased synthesis of ATP followed by cell death. © 2012 Elsevier Ltd.

Efeitos da abamectina na bioenergética de mitocôndrias isoladas de fígado de rato: Juliana Carla Castanha Zanoli. -

Pós-graduação em Ciência Animal - FMVA

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

Neuropeptide Y acts within the rat testis to inhibit testosterone secretion

The factors that influence Leydig cell activity currently include peptides such as neuropeptide Y (NPY). In this work we investigated the ability of this compound, injected directly into the testes of adult male rats, to alter testosterone (T) release into the general circulation. At a 5μg/kg dose administered 1h prior to challenge with human chorionic gonadotropin (hCG, 1.0 U/kg, iv), NPY significantly (P<0.01) blunted the T response to this gonadotropin. The inhibitory effect of NPY was observed in animals pretreated with an antagonist to gonadotropin-releasing hormone or not, indicating that the decrease in plasma T found was most likely independent of pituitary luteinizing hormone. However, testicular levels of steroidogenic acute regulatory (STAR) protein or translocator protein (TSPO) in the Leydig cells did not exhibit consistent changes, which suggested that other mechanisms mediated the blunted T response to hCG. We therefore used autoradiography and immunohistochemistry methodologies to identify NPY receptors in the testes, and found them primarily located on blood vessels. Competition studies further identified these receptors as being Y(1), a subtype previously reported to modulate the vasoconstrictor effect of NPY. The absence of significant changes in STAR and TSPO levels, as well as the absence of Y(1) receptors on Leydig cells, suggest that NPY-induced decreases in T release is unlikely to represent a direct effect of NPY on these cells. Rather, the very high expression levels of Y(1) found in testicular vessels supports the concept that NPY may alter gonadal activity, at least in part, through local vascular impairment of gonadotropin delivery to, and/or blunted T secretion from, Leydig cells.

Expression of zebra fish aromatase cyp19a and cyp19b genes in response to the ligands of estrogen receptor and aryl hydrocarbon receptor

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17beta-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERalpha, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligands.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial -barrel proteins specialized in mitochondrial genome inheritance

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum–mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA–cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

Translocation of inserted foreign epitopes by a channelforming protein

Certain bacterial protein toxins are able to insert themselves into, and at least partially across, lipid bilayer membranes in the absence of any auxiliary proteins, by using unknown mechanisms to overcome the high energy barrier presented by the hydrophobic bilayer core. We have previously shown that one such toxin, colicin Ia, translocates a large, hydrophilic part of itself completely across a lipid bilayer in conjunction with the formation of an ion-conducting channel. To address the question of whether the colicin can translocate any arbitrary amino acid sequence, we have altered the translocated segment by inserting, singly, two different foreign epitopes. Colicins containing either epitope retain significant bactericidal activity and form channels of normal conductance in planar bilayers. Furthermore, antibodies added on the side of the bilayer opposite that to which the colicin was added interact specifically with the corresponding epitopes, producing an inhibition of channel closing. Thus, the inserted epitopes are translocated along with the rest of the segment, suggesting that a surprisingly small part of colicin Ia, located elsewhere in the molecule, acts as a nonspecific protein translocator.

A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.

Oxaloacetate Transport into Plant Mitochondria1

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.

Cloning and characterization of KAP3: a novel kinesin superfamily-associated protein of KIF3A/3B.

We previously reported that KIF3A and KIF3B form a heterodimer that functions as a microtubule-based fast anterograde translocator of membranous organelles. We have also shown that this KIF3A/3B forms a complex with other associated polypeptides, named kinesin superfamily-associated protein 3 (KAP3). In the present study, we purified KAP3 protein by immunoprecipitation using anti-KIF3B antibody from mouse testis. Microsequencing was carried out, and we cloned the full-length KAP3 cDNA from a mouse brain cDNA library. Two isoforms of KAP3 exist [KAP3A (793 aa) and KAP3B (772 aa)], generated by alternative splicing in the carboxyl terminus region. Their amino acid sequences have no homology with those of any other known proteins, and prediction of their secondary structure indicated that almost the entire KAP3 molecule is alpha-helical. We produced recombinant KAP3 and KIF3A/3B using a baculovirus-Sf9 expression system. A reconstruction study in Sf9 cells revealed that KAP3 is a globular protein that binds to the tail domain of KIF3A/3B. The immunolocalization pattern of KAP3 was similar to that of KIF3A/3B in nerve cells. In addition, we found that KAP3 does not affect the motor activity of KIF3A/3B. KAP3 was associated with a membrane-bound form of KIF3A/3B in a fractional immunoprecipitation experiment, and since the KIF3 complex was found to bind to membranous organelles in an EM study, KAP3 may regulate membrane binding of the KIF3 complex.

Effects of copper on the photosynthesis of intact chloroplasts: interaction with manganese

Highly purified, intact chloroplasts were prepared from pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) following an identical procedure, and were used to investigate the cupric cation inhibition on the photosynthetic activity. In both species, copper inhibition showed a similar inhibitor concentration that decreases the enzyme activity by 50% (IC(50) approximately 1.8 microM) and did not depend on the internal or external phosphate (Pi) concentration, indicating that copper did not interact with the Pi translocator. Fluorescence analysis suggested that the presence of copper did not facilitate photoinhibition, because there were no changes in maximal fluorescence (F(m)) nor in basal fluorescence (F(o)) of copper-treated samples. The electron transport through the photosystem II (PSII) was also not affected (operating efficiency of PSII-F'v/F'm similar in all conditions). Yet, under Cu(2+) stress, the proportion of open PSII reaction centers was dramatically decreased, and the first quinone acceptor (Q(A)) reoxidation was fully inhibited, as demonstrated by the constant photochemical quenching (q(P)) along experiment time. The quantum yield of PSII electron transport (Phi(PSII)) was also clearly affected by copper, and therefore reduced the photochemistry efficiency. Manganese, when added simultaneously with copper, delayed the inhibition, as measured by oxygen evolution and chlorophyll fluorescence, but neither reversed the copper effect when added to copper-inhibited plastids, nor prevented the inhibition of the Hill activity of isolated copper-treated thylakoids. Our results suggest that manganese competed with copper to penetrate the chloroplast envelope. This competition seems to be specific because other divalent cations e.g. magnesium and calcium, did not interfere with the copper action in intact chloroplasts. All results do suggest that, under these conditions, the stroma proteins, such as the Calvin-Benson cycle enzymes or others are the most probable first target for the Cu(2+) action, resulting in the total inhibition of chloroplast photosynthesis and in the consequent unbalanced rate of production and consumption of the reducing power.