Biomedical subjects
M M Peña
Publications and source records attributed to M M Peña.
Biodegradation of phenol in a continuous process: comparative study of stirred tank and fluidized-bed bioreactors.
The paper presents the main results obtained from the study of the biodegradation process of phenol by a pure culture of Pseudomonas putida ATCC 17484. The experimental work was carried out in two different systems: a stirred tank where cells grew as a suspended culture and a fluidized bed where cells were immobilized within calcium alginate gel beads. The influence of the hydraulic residence time (HRT) and organic loading rate on the removal efficiency of phenol was determined for both bioreactors. Also, the stability of the fluidized-bed bioreactor (FBB) in terms of its ability to withstand sudden phenol overdoses is also reported. Experimental values indicated that both bioreactors showed high phenol degradation efficiencies, higher than 90%, even for a phenol loading rate in the influent as high as 4 g phenol/l day. The FBB showed better performance than the suspended-culture bioreactor due to its better control and because it could operate with lower HRT.
A copper-sensing transcription factor regulates iron uptake genes in Schizosaccharomyces pombe.
Copper and iron serve essential functions as catalytic co-factors in a wide variety of critical cellular enzymes. Studies in yeast have demonstrated an absolute dependence upon copper acquisition for proper assembly and function of the iron transport machinery. We have cloned genes for a high affinity copper transporter (Ctr4) and copper-sensing transcription factor (Cuf1) from Schizosaccharomyces pombe. Interestingly, the primary structure of Ctr4 and a putative human high affinity copper transport protein, hCtr1, suggests that they are derived from a fusion of the functionally redundant but structurally distinct Ctr1 and Ctr3 copper transporters from Saccharomyces cerevisiae. Furthermore, although Cuf1 activates ctr4(+) gene expression under copper starvation conditions, under these same conditions Cuf1 directly represses expression of genes encoding components of the iron transport machinery. These studies have identified an evolutionary step in which copper transport modules have been fused, and describe a mechanism by which a copper-sensing factor directly represses expression of the iron uptake genes under conditions in which the essential copper co-factor is scarce.
A delicate balance: homeostatic control of copper uptake and distribution.
The cellular uptake and intracellular distribution of the essential but highly toxic nutrient, copper, is a precisely orchestrated process. Copper homeostasis is coordinated by several proteins to ensure that it is delivered to specific subcellular compartments and copper-requiring proteins without releasing free copper ions that will cause damage to cellular components. Genetic studies in prokaryotic organisms and yeast have identified membrane-associated proteins that mediate the uptake or export of copper from cells. Within cells, small cytosolic proteins, called copper chaperones, have been identified that bind copper ions and deliver them to specific compartments and copper-requiring proteins. The identification of mammalian homologues of these proteins reveal a remarkable structural and functional conservation of copper metabolism between bacteria, yeast and humans. Furthermore, studies on the function and localization of the products of the Menkes and Wilson's disease genes, which are defective in patients afflicted with these diseases, have provided valuable insight into the mechanisms of copper balance and their role in maintaining appropriate copper distribution in mammals.
Copper differentially regulates the activity and degradation of yeast Mac1 transcription factor.
Copper is an essential metal ion that is toxic when accumulated to high intracellular concentrations. The yeast Mac1 protein is a copper-sensing transcription factor that is essential for both the activation and inactivation of genes required for high affinity copper ion transport. Here we demonstrate that in response to low copper ion concentrations Mac1 protein is rendered inactive for copper transporter gene transcription. Under high copper ion concentrations Mac1 is degraded in a rapid, copper-specific manner. This degradation is critical to prevent copper toxicity that would otherwise result from sustained expression of the copper transport genes. These results demonstrate that nutritional and toxic copper concentrations elicit distinct fates for the Mac1 copper-sensing transcription factor and establish a new mechanism by which trace metals regulate gene expression.
Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae.
The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to copper. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient. CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain. In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression. Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity. These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.
Copper-binding motifs in catalysis, transport, detoxification and signaling.
Copper is required for many biological processes but is toxic at high cellular concentrations, so levels in the cell must be strictly controlled. Copper-binding motifs have been identified and characterized in many proteins. The way in which copper is coordinated by these motifs is important for the transport and distribution of intracellular copper and for the effective functioning of copper-dependent enzymes.
The DpsA protein of Synechococcus sp. Strain PCC7942 is a DNA-binding hemoprotein. Linkage of the Dps and bacterioferritin protein families.
The Dps family of proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long term nutrient limitation. Some members of the Dps protein family have been shown to form large (approximately 150-kDa), hexameric complexes that bind chromosomal DNA with little sequence specificity. In this paper we report the nucleotide sequence of the dpsA gene from Synechococcus sp. PCC7942 encoding a cyanobacterial Dps homolog. The deduced amino acid sequence of the Synechococcus sp. DpsA protein revealed that a carboxyl-terminal domain of the protein was > 60% homologous to the COOH-terminal half of bacterioferritin. Other known Dps family members lack such high similarity to the bacterioferritins. Purification and spectroscopic analysis of the Synechococcus sp. DpsA protein complex revealed that the complex contains heme and has a weak catalase activity in vitro. Activity staining of nondenaturing polyacrylamide gels showed that the protein complex comigrated with both the heme and the catalase activity, and O2 evolution measurements yielded a maximal specific activity of 1.7 mumol of H2O2 consumed/micrograms of protein-1 min-1. We speculate that the protein may have a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggest that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Last, the evolutionary link between the Dps protein family and the bacterioferritins is discussed.
Purification and characterization of a Synechococcus sp. strain PCC 7942 polypeptide structurally similar to the stress-induced Dps/PexB protein of Escherichia coli.
A stable DNA/protein complex having an apparent molecular mass of approximately 150 kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.