Copper��Dopamine Complex Induces Mitochondrial Autophagy Preceding Caspase-independent Apoptotic Cell Death

Parkinsonism is one of the major neurological symptoms in Wilson disease, and young workers who worked in the copper smelting industry also developed Parkinsonism. We have reported the specific neurotoxic action of copper·dopamine complex in neurons with dopamine uptake. Copper·dopamine complex (100 μm) induces cell death in RCSN-3 cells by disrupting the cellular redox state, as demonstrated by a 1.9-fold increase in oxidized glutathione levels and a 56% cell death inhibition in the presence of 500 μm ascorbic acid; disruption of mitochondrial membrane potential with a spherical shape and well preserved morphology determined by transmission electron microscopy; inhibition (72%, p < 0.001) of phosphatidylserine externalization with 5 μm cyclosporine A; lack of caspase-3 activation; formation of autophagic vacuoles containing mitochondria after 2 h; transfection of cells with green fluorescent protein-light chain 3 plasmid showing that 68% of cells presented autophagosome vacuoles; colocalization of positive staining for green fluorescent protein-light chain 3 and Rhod-2AM, a selective indicator of mitochondrial calcium; and DNA laddering after 12-h incubation. These results suggest that the copper·dopamine complex induces mitochondrial autophagy followed by caspase-3-independent apoptotic cell death. However, a different cell death mechanism was observed when 100 μm copper·dopamine complex was incubated in the presence of 100 μm dicoumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (EC 1.6.99.2, also known as DT-diaphorase and NQ01), because a more extensive and rapid cell death was observed. In addition, cyclosporine A had no effect on phosphatidylserine externalization, significant portions of compact chromatin were observed within a vacuolated nuclear membrane, DNA laddering was less pronounced, the mitochondria morphology was more affected, and the number of cells with autophagic vacuoles was a near 4-fold less.A possible role of copper in the neurodegeneration of dopaminergic neurons is supported by the fact that patients with neurological Wilson disease, a copper deposition disorder, display a number of extrapyramidal motor symptoms, including Parkinsonism. The cerebral manifestations in neurological Wilson disease are expressed as bradykinesia, rigidity, tremor, dyskinesia, and dysarthria (1). It has been proposed that neurological Wilson disease can be assigned to the group of secondary Parkinsonian syndromes (2). Interestingly, young workers who worked in the copper smelting industry also developed Parkinsonism (3).Studies performed in rats showed copper (Cu²⁺)-induced degeneration of dopaminergic neurons in the nigrostriatal system. Likewise, it was described that copper neurotoxicity in rat substantia nigra and striatum is dependent on NAD(P)H dehydrogenase inhibition (4, 5). All of these results support a possible role for copper in the neurodegeneration of dopaminergic neurons.The general mechanism of toxicity, induced by the reduced form of copper (Cu⁺) catalyzing the formation of hydroxyl radicals in the presence of hydrogen peroxide through the Fenton reaction, cannot explain the specific degeneration of dopaminergic neurons in Parkinsonism induced in neurological Wilson disease, or in miners working in the copper smelting industry. The selective action of copper can be explained by the ability of copper to form a complex with dopamine, allowing this metal to be transported by cells that have the ability to take up dopamine (6). This specific neurotoxic action of copper in neurons with dopamine uptake is dependent on (i) the ability of copper to form a complex with dopamine (Cu·DA)² (6, 7), (ii) uptake of Cu·DA complex by dopamine transporters, (iii) oxidation of dopamine to aminochrome, and (iv) one-electron reduction of aminochrome by inhibiting NAD(P)H dehydrogenase (6). These findings may explain the selective neurotoxic action of copper in the brain, but they do not explain the cell death mechanism.Currently, cell death is divided into three categories: apoptosis, autophagy, and necrosis. At the current time, only apoptosis and autophagic cell death are generally accepted as being legitimate forms of programmed cell death. Alternative models of cell death have therefore been proposed, including para-apoptosis, mitotic catastrophe, oncosis, and pyroptosis (8–12). Necrosis is characterized mostly by the absence of caspase activation, cytochrome c release, and DNA oligonucleosomal fragmentation. Apoptotic cells are characterized by the formation of blebs, chromatin condensation, DNA oligonucleosomal fragmentation, and exposure of phosphatidylserine on the external membrane. This mode of cell death can be dependent or independent of activation of caspases (13). On the other hand, autophagy can be distinguished from apoptosis by sequestration of bulk cytoplasm and organelles in double or multimembrane autophagic vacuoles that then fuse with the lysosomal system. Some of these described mechanisms are related to neurological diseases such as Parkinson disease (14, 15). Cells can use different methods to activate their own destruction, and more than one death program may be activated at the same time (16, 17).The purpose of this study was to examine the Cu·DA complex-induced cell death mechanism in RCSN-3 cells, a cell line that expresses dopamine, norepinephrine, and serotonin transporters (18, 19).     

Phylogeographical structure of the neotropical forest tree Hymenaea courbaril (Leguminosae: Caesalpinioideae) and its relationship with the Vicariant Hymenaea stigonocarpa from Cerrado

The phylogeography of Hymenaea courbaril var. stilbocarpa from Atlantic Forest and riverine forests of the Cerrado biome in central and southeastern Brazil was investigated. The data were compared with those of its congeneric Hymenaea stigonocarpa, a typical tree from savanna. In the Cerrado, H. courbaril var. stilbocarpa is found in sites contiguous with those of H. stigonocarpa, and they share common life-history attributes. The psbC/trnS3 region of the chloroplast DNA was sequenced in 149 individuals of H. courbaril var. stilbocarpa. High genetic variation was found in this species, with the identification of 18 haplotypes, similarly to what was found in H. stigonocarpa with 23 haplotypes in the same geographic region. Populations of H. courbaril var. stilbocarpa could be structured in 3 phylogeographic groups. Spatial analysis of molecular variation indicated that 46.4% of the genetic variation was due to differences among these groups. Three haplotypes were shared by H. courbaril var. stilbocarpa and H. stigonocarpa, and only 10.5% of the total genetic variation could be attributed to between-species difference. We surmise that during the glacial times, H. courbaril var. stilbocarpa populations must have gone extinct in most parts of the southern of its present-day occurrence area. After climate amelioration, these areas were probably recolonized from northern and eastern. The relatively similar phylogeographic structure of vicariant Hymenaea species suggests that they were subjected to the same impacts during the Quaternary climatic fluctuations. The sharing of haplotypes and the genetic similarity between the 2 Hymenaea species suggest the existence of ancestral polymorphism and/or hybridization.     

Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei

Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZα gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-P_BAD-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Δasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.Burkholderia pseudomallei is the etiologic agent of melioidosis (3, 35). While the bacterium and disease are typically endemic to tropical and subtropical regions of the world (5), historical precedent for use in bioweapon development programs, low infectious doses, high morbidity and mortality, and arduous therapy caused B. pseudomallei to be listed as a category B select agent by the Centers for Disease Control and Prevention. In the United States, transport, possession, and use of select agents is regulated by strict federal guidelines. These guidelines restrict the use of antibiotic resistance markers in research to those that do not compromise the use of the respective drugs in humans, veterinary medicine, or agriculture (27). The paucity of selection markers approved for this bacterium has led to development of genetic manipulation strategies that allow the isolation of unmarked mutants. These include fragment mutagenesis, where a linear DNA fragment containing the mutation, assembled in vitro by PCR, is transferred to the host strain and selection for the antibiotic resistance encoded by the fragment results in gene replacement in the homologous region of the chromosome (4, 32). When the selection markers are flanked by Cre or Flp recombinase target sites, they can be removed in vivo by temporary expression of the respective site-specific recombinase, resulting in markerless mutants (4).Additionally, allelic replacement schemes driven by genetically engineered pheS- (1, 20), sacB- (9, 15), and rpsL- (19, 30) based counterselection markers have been developed for use in B. pseudomallei. With these technologies, regions of homology containing markerless mutations are cloned into a nonreplicative plasmid. Transfer of the recombinant plasmid into the bacterial host followed by selection of an antibiotic resistance encoded by a gene located on the plasmid backbone leads to integration of the nonreplicative plasmid by regions of homology. Loss of plasmid sequences by homologous recombination results in a population in which a significant portion of the survivors of the appropriate counterselection will have undergone the desired gene replacement (Fig. ​(Fig.1,1, lower right).Open in a separate windowFIG. 1.Schematic of allele exchange procedures. For plasmid-based allelic exchange, a PCR-assembled chromosomal segment containing a deletion of orfY with flanking orfX and orfZ sequences is cloned into an appropriate vector, e.g., pEXKm5. The nonreplicative plasmid is delivered to the host strain by conjugation (or electroporation), followed by kanamycin resistance selection. This step results in integration of the allelic replacement construct into the chromosome by homologous recombination between cloned and chromosomal sequences and can be visualized by the appearance of blue colonies on Km- and X-Gluc-containing medium. The two different merodiploid resolution strategies enabled by pEXKm5 are illustrated. For I-SceI-catalyzed resolution (illustrated on the left side), the merodiploid is transformed with the I-SceI expression construct, which results in double-stranded cleavage of the chromosome and release of most of the plasmid backbone. This event can be monitored by the appearance of white colonies on X-Gluc-containing medium. Repair of the double-stranded break by homologous flanking repeat sequences leads to formation of a wild-type strain (event denoted by the circled number 1) or a mutant strain (event denoted by the circled number 2). The two events are distinguished by phenotypic analyses and/or PCR. In a final step that is not illustrated in this figure, purification of colonies with the desired mutant genotype/phenotype at 42°C leads to loss of the pBADSce expression vector in 100% of the colonies. For sacB-mediated counterselection (illustrated on the right side), the merodiploid strain is plated on medium containing sucrose. This counterselection will either result in a wild-type strain (event denoted by Δ1) or in a mutant strain (event denoted by Δ2). These events can be monitored by the appearance of white colonies on X-Gluc- and sucrose-containing medium and are distinguished by phenotypic analyses and/or PCR. Abbreviations: gusA, Escherichia coli glucuronidase-encoding gene; ori, pMB9-derived narrow-range origin of replication; sacB, Bacillus subtilis levansucrase-encoding gene optimized for expression and localization in B. pseudomallei (9).An alternative to counterselection schemes involves the use of the intron-encoded homing endonuclease I-SceI (18). This enzyme recognizes a specific 18-bp sequence which is absent from all eukaryotic genomes (except the source, Saccharomyces cerevisiae) and prokaryotic genomes sequenced to date. The basic principle of the method is that cleavage of the bacterial chromosome at an artificially introduced I-SceI site(s) stimulates recombination (21). In this scheme, a nonreplicative plasmid containing cloned regions of homology and an I-SceI site(s) is integrated into the chromosome by homologous recombination between cloned and chromosomal sequences. This integration event is selected by an antibiotic resistance encoded by a gene on the plasmid backbone and results in merodiploid formation. Next, expression of the I-SceI enzyme, either encoded by the integrated plasmid or by a separately introduced plasmid, results in cleavage at the I-SceI site(s) within the integrated vector sequences (Fig. ​(Fig.1,1, lower left). The resulting double-strand break is repaired by the host recombination machinery by recombination of the regions of sequence homology flanking the break in the merodiploid. Loss of plasmid sequences by homologous recombination results in a mixed population in which a certain percentage will have undergone the desired gene replacement, given the gene is nonessential under the experimental conditions. Although cleavage of the chromosome induces the host''s SOS response, this does not result in an increased mutation rate (21). The use of I-SceI for promoting allelic exchange in Escherichia coli (21), Bacillus anthracis (11), Burkholderia cenocepacia (8), Corynebacterium glutamicum (31), and Pseudomonas aeruginosa (36) has been reported.Biparental or triparental mating is the most efficient way to introduce nonreplicative plasmids into B. pseudomallei for purposes of allele replacement. An obvious disadvantage of this method is that a donor strain must be available that is compatible with the conjugative plasmid, e.g., it cannot contain antibiotic resistance markers that interfere with those encoded by the conjugative plasmid. And herein lie the problems. The most commonly used E. coli mobilizer strains, S17-1 (29) and SM10 (29) and its derivatives, e.g., SM10(λpir) (17), contain chromosomally integrated RP4 sequences and, therefore, different resistance markers, depending on how they were engineered. For example, the most versatile mobilizer strain, SM10(λpir), is kanamycin resistant (Km^r) and can therefore not be used with genetic elements containing an nptII Km^r-encoding gene, one of the few approved selection markers that work with all clinical and environmental B. pseudomallei strains tested to date. Conjugation experiments require counterselection against the donor and untransformed recipient strains. This is usually achieved by utilizing an antibiotic or growth medium that precludes growth of the donor strain but does not affect the recipient strain. For instance, E. coli is highly susceptible to polymyxin B but Burkholderia spp. are naturally resistant to this antimicrobial. Similarly, Pseudomonas aeruginosa can utilize citrate but E. coli cannot. To avoid the use of antibiotics or for situations where other intrinsic properties cannot be exploited, donor strains have been engineered that require nutritional supplements for growth, but they either still contain antibiotic resistance markers or are based on nutritional requirements that preclude the use of rich media (1, 6).Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a single vector which contains the features required for resolution of merodiploids using either I-SceI-driven recombination or sacB-based counterselection. Complementing this system is an improved E. coli mobilizer strain susceptible to all antibiotics and counterselectable on rich media. The versatility of these methods was demonstrated by isolation of deletion mutants as well as a temperature-sensitive allele in an essential fatty acid biosynthesis gene.     

Neurotoxin Gene Clusters in Clostridium botulinum Type Ab Strains

There is limited knowledge of the neurotoxin gene diversity among Clostridium botulinum type Ab strains. Only the sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene in C. botulinum type Ab strain CDC588 have been reported. In this study, we sequenced the entire bont/A- and bont/B-associated neurotoxin gene clusters of C. botulinum type Ab strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 and the bont/A gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The bont/A nucleotide sequence of strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 differed from those of the known bont/A subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The bont/B nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 showed 99.7% identity to the sequence of subtype B1. The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the C. botulinum type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of p21-orfX1 and orfX2-orfX3 varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and bont/A and bont/B nucleotide sequences, suggesting that they may have arisen from separate recombination events.Clostridium botulinum is a gram-positive anaerobic bacterium that produces an extremely potent toxin, the botulinum neurotoxin (BoNT). There are seven serologically distinct types of BoNT (serotypes A through G). Although most strains of C. botulinum express a single toxin serotype, some isolates have been shown to produce more than one, namely, Ab, Af, Ba, and Bf (11). In addition, many strains designated type A by mouse bioassay harbor nucleotide sequences for both type A and B toxins (6). These strains have been designated A(B) to indicate the presence of the bont/B gene without type B-specific toxicity.Based on phylogenetic analysis of the neurotoxin gene sequences, four subtypes have been identified within serotype A and five subtypes within serotype B (12). The neurotoxin gene nucleotide sequences of these subtypes differ by up to 8%, and the predicted amino acid sequences differ by up to 16%. In addition, the genes encoding components of the toxin complexes are arranged in clusters that differ in composition and organization (14) (Fig. ​(Fig.1).1). The toxin gene cluster of subtype A1 (termed ha cluster) includes the gene encoding the nontoxic nonhemagglutinin (ntnh), a regulatory gene (botR), and an operon encoding three hemagglutinins (ha70, ha33, and ha17). The toxin gene clusters containing bont/A2 or bont/A3 (termed orfX cluster) include the ntnh and p21 (analogous to botR) genes and several genes of unknown function (orfX1, orfX2, orfX3, and p47). Type Ba and A(B) strains contain two sets of neurotoxin cluster genes in which ha70, ha33, and ha17 are associated with the bont/B gene, and orfX1, orfX2, orfX3, and p47 are associated with the bont/A gene. In addition, some A1 strains contain a neurotoxin gene cluster that is similar to those in A2 and A3, but the bont/A nucleotide sequence is 99.9% identical to that in other A1 strains. These strains have been designated HA⁻ Orfx⁺ A1 (14). The neurotoxin gene cluster in type B strains includes the ntnh, botR, ha70, ha33, and ha17 genes. Notably, no differences in the neurotoxin gene cluster arrangements among the subtypes within serotype B have been reported.Open in a separate windowFIG. 1.Toxin gene cluster arrangements for BoNT type A-producing strains, including Ab, A(B), and Ba strains.Although several studies have described the organization and the nucleotide sequences of the neurotoxin gene cluster components among type A and B strains [including type Ba and A(B) strains], there is limited information regarding the diversity of the neurotoxin cluster genes among C. botulinum type Ab strains. The nucleotide sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene of C. botulinum type Ab strain CDC588 have been previously reported; strain CDC1436 harbors a bont/A2 gene, and both strains CDC1436 and CDC588 harbor a bont/bvB gene (12, 15). Four additional type Ab strains from Italy have been analyzed by a restriction fragment length polymorphism method to determine the bont/A and bont/B subtypes (7, 9). To the best of our knowledge, the complete nucleotide sequences of the neurotoxin gene clusters in C. botulinum type Ab strains have not been reported. Thus, the objective of this study was to analyze the neurotoxin gene cluster composition in three C. botulinum type Ab strains ({"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, CDC588, and CDC1436) available in the CDC strain collection. We report differences in bont/A gene sequence among type Ab strains, including the identification of a novel bont/A nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, and describe differences in the organization of the neurotoxin gene clusters among these strains.     

Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium

Cadmium (Cd) is a toxic heavy metal, which can cause severe damage to plant development. The aim of this work was to characterize ultrastructural changes induced by Cd in miniature tomato cultivar Micro-Tom (MT) mutants and their wild-type counterpart. Leaves of diageotropica (dgt) and Never ripe (Nr) tomato hormonal mutants and wild-type MT were analysed by light, scanning and transmission electron microscopy in order to characterize the structural changes caused by the exposure to 1 mM CdCl₂. The effect of Cd on leaf ultrastructure was observed most noticeably in the chloroplasts, which exhibited changes in organelle shape and internal organization, of the thylakoid membranes and stroma. Cd caused an increase in the intercellular spaces in Nr leaves, but a decrease in the intercellular spaces in dgt leaves, as well as a decrease in the size of mesophyll cells in the mutants. Roots of the tomato hormonal mutants, when analysed by light microscopy, exhibited alterations in root diameter and disintegration of the epidermis and the external layers of the cortex. A comparative analysis has allowed the identification of specific Cd-induced ultrastructural changes in wild-type tomato, the pattern of which was not always exhibited by the mutants.     

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.     

Genomic and functional characterization of StCDPK1

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4–8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (β7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the β7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.