Alejandro Yañez - Academia.edu (original) (raw)

Papers by Alejandro Yañez

Molecular aspects of medicine, Jun 18, 2017

Adenosine is a nucleoside that is particularly interesting to many scientific and clinical commun... more Adenosine is a nucleoside that is particularly interesting to many scientific and clinical communities as it has important physiological and pathophysiological roles in the kidney. The distribution of adenosine receptors has only recently been elucidated; therefore it is likely that more biological roles of this nucleoside will be unveiled in the near future. Since the discovery of the involvement of adenosine in renal vasoconstriction and regulation of local renin production, further evidence has shown that adenosine signaling is also involved in the tubuloglomerular feedback mechanism, sodium reabsorption and the adaptive response to acute insults, such as ischemia. However, the most interesting finding was the increased adenosine levels in chronic kidney diseases such as diabetic nephropathy and also in non-diabetic animal models of renal fibrosis. When adenosine is chronically increased its signaling via the adenosine receptors may change, switching to a state that induces renal...

Journal of cellular physiology, Jan 17, 2016

Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (Na... more Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti-diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type-2-diabetic (D-RPTEC) and non-diabetic (N-RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development and repair, all processes that are dysregulated during DKD. In basal conditions, D-RPTEC cells secreted higher levels of prototypical pro-inflammatory IL-6, IL-8 and MCP-1 than N-RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL-6, IL-8 and MCP-1 secretion in both N- and D-RPTEC, together with lower levels of IL-1 RA, IL-4, IL-10 and GM-CSF, suggesting that it may contribute to the extent of renal damage/repair ...

Biochemical Journal, 2015

Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the ... more Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein–protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein–protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, ...

Reactive and Functional Polymers, 2014

Immobilization of rhodamine 6G in calcium alginate microcapsules was achieved using the polyanion... more Immobilization of rhodamine 6G in calcium alginate microcapsules was achieved using the polyanion bearing negatively charged aromatic groups poly(sodium 4-styrenesulfonate) as complexing agent. The immobilization of the dye by this method finds its basis on the stabilization of the dye/polymer complex by short-range aromatic-aromatic interactions, which are resistant to the cleaving effect of highly concentrated electrolytes. On the contrary, direct immobilization of the dye in the microcapsules resulted unsuccessful due to its high diffusion coefficient in the aqueous medium, and complexation with poly(sodium vinylsulfonate) did not improve the immobilization, since the corresponding complex is based on long-range electrostatic interactions, which are easily cleaved under the high ionic strength conditions of the microcapsule formation reaction. Thus, the present investigation represents a proof of concept on the use of aromatic-aromatic interactions between polyelectrolytes bearing charged aromatic rings and their aromatic counterions as a tool to achieve improved functionalities. The release of the rhodamine 6G/poly(sodium 4-styrenesulfonate) complex from the microcapsules has been investigated as a function of pH and temperature. Coating the microcapsules with chitosan allowed minimizing the release of the dye from the microcapsules.

Kidney International, 2008

Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters t... more Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters that mediate high affinity sodium-dependent L-ascorbic acid transport. Sodium-vitamin C cotransporter-1 mRNA has been detected in intestine and liver and the S3 segment of the renal proximal tubule. Here, we found that its distribution was broader and all three proximal tubule segments of mouse and human expressed the transporter but the S3 segment had the highest expression. Sodium-vitamin C co-transporter-1 expression was also found in the renal epithelial-derived LLC-PK1 cell line. Ascorbic acid transport in these cells was regulated by a single kinetic component that depended on the sodium concentration, pH and temperature. Reducing ascorbate concentration increased the apical expression of the transporter suggesting the presence of a feedback system for regulation of transporter abundance at the luminal membrane.

Journal of Biological Chemistry, 1998

1 The abbreviations used are: mAAT, mitochondrial aspartate aminotransferase; cAAT, cytosolic asp... more 1 The abbreviations used are: mAAT, mitochondrial aspartate aminotransferase; cAAT, cytosolic aspartate aminotransferase; pmAAT, precursor to aspartate aminotransferase; pcAAT, precursor chimera to cytosolic aspartate aminotransferase; pcmAAT, chimeric precursor; ⌬, membrane potential; mt-hsp70, mitochondrial hsp70; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; RRL, rabbit reticulocyte lysate; TPCK, L-1-tosylamido-2-phenylethyl chloromethylketone; HEPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.

FEBS Letters, 2004

In primary cultured hepatocytes, fructose-1,6-bisphosphatase (FBPase) localization is modulated b... more In primary cultured hepatocytes, fructose-1,6-bisphosphatase (FBPase) localization is modulated by glucose, dihydroxyacetone (DHA) and insulin. In the absence of these substrates, FBPase was present in the cytoplasm, but the addition of glucose or DHA induced its translocation to the nucleus. As expected, we observed the opposite effect of glucose on glucokinase localization. The addition of insulin in the absence of glucose largely increased the amount of nuclear FBPase. Moreover, at high concentrations of glucose or DHA, FBPase shifted from the cytosol to the cell periphery and co-localized with GS. Interestingly, the synthesis of Glu-6-P and glycogen induced by DHA was not inhibited by insulin. These results indicate that FBPase is involved in glycogen synthesis from gluconeogenic precursors. Overall, these findings show that translocation may be a new integrative mechanism for gluconeogenesis and glyconeogenesis.

FEBS Letters, 2003

Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzym... more Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzymes. We report our findings on the intracellular localization of liver FBPase in rat liver and kidney, the main organs in the endogenous glucose production. Immunofluorescence and confocal analysis revealed that FBPase was present in the cytosol and, unexpectedly, inside the nucleus of hepatocytes and proximal cells of the nephron. Additionally, FBPase was found in the plasma membrane area of adjacent hepatocytes where glycogen is synthesized and in the apical region of proximal kidney cells. This subcellular distribution in multiple compartments suggests the presence of different localization signals on FBPase for diverse metabolic functions.

Biology of Reproduction, 2004

In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metab... more In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [ 14 C]glycogen after the addition to l-CCM with [ 14 C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2014

Background: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, ... more Background: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P 2 and by high concentrations of its substrate Fru-1,6-P 2. The mechanism that produces substrate inhibition continues to be obscure. Methods: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P 2 and Fru-1,6-P 2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. Results: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P 2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P 2 , competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. Conclusions: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. General significance: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.

Postdoc Journal, 2017

Sodium tungstate (NaW) is an inorganic salt that has proven to be a potent insulin-mimetic agent,... more Sodium tungstate (NaW) is an inorganic salt that has proven to be a potent insulin-mimetic agent, although the molecular events seems to differ. Inhibition of hepatic gluconeogenesis is one significant physiological action of insulin, therefore studying the effect of NaW on gluconeogenic enzymes will contribute to understand its elusive mechanism of action. Here, we show that NaW has no inhibitory effect over the gluconeogenic enzyme fructose 1,6-bisphosphatase (FBPase) in vitro, but mimics insulin in the induction of the nuclear translocation of FBPase in rat liver, which may have a negative impact on its activity. Then, at least in part, NaW may inhibit hepatic gluconeogenesis by inducing the proper subcellular distribution of FBPase, without directly interfering with its phosphatase activity.

Biomedicines

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemi... more Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemia generates reactive oxygen species (ROS), contributing to diabetic complications, especially in DN. Sodium Tungstate (NaW) is an effective antidiabetic agent for short and long-term treatments of both type 1 and type 2 diabetes models. In this study, we evaluated the effect of NaW on ROS production in bovine neutrophils incubated with platelet-activating factor (PAF) and in HK-2 cells induced by high glucose or H2O2. In addition, we evaluated the effect on iNOS expression in the type 1 diabetic rat model induced with streptozotocin (STZ). NaW inhibited ROS production in PAF-induced bovine neutrophils, and human tubular cells (HK-2) were incubated in high glucose or H2O2. In addition, NaW inhibited iNOS expression in glomeruli and tubular cells in the type 1 diabetic rat. This study demonstrates a new role for NaW as an active antioxidant and its potential use in treating DN.

Polymers

The development of fish oral vaccines is of great interest to the aquaculture industry due to the... more The development of fish oral vaccines is of great interest to the aquaculture industry due to the possibility of rapid vaccination of a large number of animals at reduced cost. In a previous study, we evaluated the effect of alginate-encapsulated Piscirickettsia salmonis antigens (AEPSA) incorporated in feed, effectively enhancing the immune response in Atlantic salmon (Salmo salar). In this study, we seek to characterize AEPSA produced by ionic gelation using an aerodynamically assisted jetting (AAJ) system, to optimize microencapsulation efficiency (EE%), to assess microparticle stability against environmental (pH, salinity and temperature) and gastrointestinal conditions, and to evaluate microparticle incorporation in fish feed pellets through micro-CT-scanning. The AAJ system was effective in obtaining small microparticles (d < 20 μm) with a high EE% (97.92%). Environmental conditions (pH, salinity and temperature) generated instability in the microparticles, triggering prote...

Frontiers in Immunology, 2021

An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for s... more An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1β and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only th...

Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosi... more Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosis in salmonids farms in Chile. It was recently reported that P. salmonis produces exotoxins that play a role in the pathogenesis. However a delivery system has not yet been identified. Outer membrane vesicles (OMVs) are 10-300 nm spherical-bilayer structures discharged from the surface of many Gram-negative bacteria, which are able to deliver toxins and virulence factors. Recently, we described the production of OMVs by P. salmonis in both, in normal growth in broth and inside the host cells after infection. However, pro-immflamatory cytokines expression induced by P. salmonis OMVs has not yet been characterized. Thus, the aim of this study was to investigate if OMVs purified from P. salmonis are able to generate an immflamatory immune response in fish macrophages. P. salmonis was grown in basal broth supplemented with Cysteine (3.18 mM) and ferric chloride (0.05 mM) at 18°C until early ...

Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the et... more Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the etiologic agent of Piscirickettsiosis, a systemic infection of multiple organs and tissues among as kidney, liver, spleen, brain, intestine, ovaries, and gills in several salmonids species such as Rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch), causing high mortality and serious economic losses for fish farming in the south of Chile. P. salmonis is able to infect, evade the immune response and replicate inside of several fish cell lines such as RTS-11, SHK-1 and CHSE-214. However, there are no reports concerning the infection and lifestyle of P. salmonis inside SHK-1 cell line. Thus, the aim of this study was to characterize, in a time-course, the infection and intracellular lifestyle of P. salmonis in SHK-1 fish cell line. SHK-1 cells were cultured at 20oC in 75 cm flask, in Leibovitz’s L-15 medium supplemented with 10% FBS. P. sa...

Microorganisms, 2020

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with... more Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and ses...

Scientific Reports, 2020

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-... more Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-economic impacts for Chilean salmonid aquaculture. The identification of major environmental reservoirs for P. salmonis has long been ignored. Most microbial life occurs in biofilms, with possible implications in disease outbreaks as pathogen seed banks. Herein, we report on an in vitro analysis of biofilm formation by P. salmonis Psal-103 (LF-89-like genotype) and Psal-104 (EM-90-like genotype), the aim of which was to gain new insights into the ecological role of biofilms using multiple approaches. The cytotoxic response of the salmon head kidney cell line to P. salmonis showed interisolate differences, depending on the source of the bacterial inoculum (biofilm or planktonic). Biofilm formation showed a variable-length lag-phase, which was associated with wider fluctuations in biofilm viability. Interisolate differences in the lag phase emerged regardless of the nutritional content of ...

Microorganisms, 2020

Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish ... more Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by...

Molecular aspects of medicine, Jun 18, 2017

Adenosine is a nucleoside that is particularly interesting to many scientific and clinical commun... more Adenosine is a nucleoside that is particularly interesting to many scientific and clinical communities as it has important physiological and pathophysiological roles in the kidney. The distribution of adenosine receptors has only recently been elucidated; therefore it is likely that more biological roles of this nucleoside will be unveiled in the near future. Since the discovery of the involvement of adenosine in renal vasoconstriction and regulation of local renin production, further evidence has shown that adenosine signaling is also involved in the tubuloglomerular feedback mechanism, sodium reabsorption and the adaptive response to acute insults, such as ischemia. However, the most interesting finding was the increased adenosine levels in chronic kidney diseases such as diabetic nephropathy and also in non-diabetic animal models of renal fibrosis. When adenosine is chronically increased its signaling via the adenosine receptors may change, switching to a state that induces renal...

Journal of cellular physiology, Jan 17, 2016

Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (Na... more Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti-diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type-2-diabetic (D-RPTEC) and non-diabetic (N-RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development and repair, all processes that are dysregulated during DKD. In basal conditions, D-RPTEC cells secreted higher levels of prototypical pro-inflammatory IL-6, IL-8 and MCP-1 than N-RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL-6, IL-8 and MCP-1 secretion in both N- and D-RPTEC, together with lower levels of IL-1 RA, IL-4, IL-10 and GM-CSF, suggesting that it may contribute to the extent of renal damage/repair ...

Biochemical Journal, 2015

Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the ... more Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein–protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein–protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, ...

Reactive and Functional Polymers, 2014

Immobilization of rhodamine 6G in calcium alginate microcapsules was achieved using the polyanion... more Immobilization of rhodamine 6G in calcium alginate microcapsules was achieved using the polyanion bearing negatively charged aromatic groups poly(sodium 4-styrenesulfonate) as complexing agent. The immobilization of the dye by this method finds its basis on the stabilization of the dye/polymer complex by short-range aromatic-aromatic interactions, which are resistant to the cleaving effect of highly concentrated electrolytes. On the contrary, direct immobilization of the dye in the microcapsules resulted unsuccessful due to its high diffusion coefficient in the aqueous medium, and complexation with poly(sodium vinylsulfonate) did not improve the immobilization, since the corresponding complex is based on long-range electrostatic interactions, which are easily cleaved under the high ionic strength conditions of the microcapsule formation reaction. Thus, the present investigation represents a proof of concept on the use of aromatic-aromatic interactions between polyelectrolytes bearing charged aromatic rings and their aromatic counterions as a tool to achieve improved functionalities. The release of the rhodamine 6G/poly(sodium 4-styrenesulfonate) complex from the microcapsules has been investigated as a function of pH and temperature. Coating the microcapsules with chitosan allowed minimizing the release of the dye from the microcapsules.

Kidney International, 2008

Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters t... more Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters that mediate high affinity sodium-dependent L-ascorbic acid transport. Sodium-vitamin C cotransporter-1 mRNA has been detected in intestine and liver and the S3 segment of the renal proximal tubule. Here, we found that its distribution was broader and all three proximal tubule segments of mouse and human expressed the transporter but the S3 segment had the highest expression. Sodium-vitamin C co-transporter-1 expression was also found in the renal epithelial-derived LLC-PK1 cell line. Ascorbic acid transport in these cells was regulated by a single kinetic component that depended on the sodium concentration, pH and temperature. Reducing ascorbate concentration increased the apical expression of the transporter suggesting the presence of a feedback system for regulation of transporter abundance at the luminal membrane.

Journal of Biological Chemistry, 1998

1 The abbreviations used are: mAAT, mitochondrial aspartate aminotransferase; cAAT, cytosolic asp... more 1 The abbreviations used are: mAAT, mitochondrial aspartate aminotransferase; cAAT, cytosolic aspartate aminotransferase; pmAAT, precursor to aspartate aminotransferase; pcAAT, precursor chimera to cytosolic aspartate aminotransferase; pcmAAT, chimeric precursor; ⌬, membrane potential; mt-hsp70, mitochondrial hsp70; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; RRL, rabbit reticulocyte lysate; TPCK, L-1-tosylamido-2-phenylethyl chloromethylketone; HEPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.

FEBS Letters, 2004

In primary cultured hepatocytes, fructose-1,6-bisphosphatase (FBPase) localization is modulated b... more In primary cultured hepatocytes, fructose-1,6-bisphosphatase (FBPase) localization is modulated by glucose, dihydroxyacetone (DHA) and insulin. In the absence of these substrates, FBPase was present in the cytoplasm, but the addition of glucose or DHA induced its translocation to the nucleus. As expected, we observed the opposite effect of glucose on glucokinase localization. The addition of insulin in the absence of glucose largely increased the amount of nuclear FBPase. Moreover, at high concentrations of glucose or DHA, FBPase shifted from the cytosol to the cell periphery and co-localized with GS. Interestingly, the synthesis of Glu-6-P and glycogen induced by DHA was not inhibited by insulin. These results indicate that FBPase is involved in glycogen synthesis from gluconeogenic precursors. Overall, these findings show that translocation may be a new integrative mechanism for gluconeogenesis and glyconeogenesis.

FEBS Letters, 2003

Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzym... more Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzymes. We report our findings on the intracellular localization of liver FBPase in rat liver and kidney, the main organs in the endogenous glucose production. Immunofluorescence and confocal analysis revealed that FBPase was present in the cytosol and, unexpectedly, inside the nucleus of hepatocytes and proximal cells of the nephron. Additionally, FBPase was found in the plasma membrane area of adjacent hepatocytes where glycogen is synthesized and in the apical region of proximal kidney cells. This subcellular distribution in multiple compartments suggests the presence of different localization signals on FBPase for diverse metabolic functions.

Biology of Reproduction, 2004

In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metab... more In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [ 14 C]glycogen after the addition to l-CCM with [ 14 C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2014

Background: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, ... more Background: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P 2 and by high concentrations of its substrate Fru-1,6-P 2. The mechanism that produces substrate inhibition continues to be obscure. Methods: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P 2 and Fru-1,6-P 2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. Results: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P 2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P 2 , competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. Conclusions: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. General significance: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.

Postdoc Journal, 2017

Sodium tungstate (NaW) is an inorganic salt that has proven to be a potent insulin-mimetic agent,... more Sodium tungstate (NaW) is an inorganic salt that has proven to be a potent insulin-mimetic agent, although the molecular events seems to differ. Inhibition of hepatic gluconeogenesis is one significant physiological action of insulin, therefore studying the effect of NaW on gluconeogenic enzymes will contribute to understand its elusive mechanism of action. Here, we show that NaW has no inhibitory effect over the gluconeogenic enzyme fructose 1,6-bisphosphatase (FBPase) in vitro, but mimics insulin in the induction of the nuclear translocation of FBPase in rat liver, which may have a negative impact on its activity. Then, at least in part, NaW may inhibit hepatic gluconeogenesis by inducing the proper subcellular distribution of FBPase, without directly interfering with its phosphatase activity.

Biomedicines

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemi... more Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemia generates reactive oxygen species (ROS), contributing to diabetic complications, especially in DN. Sodium Tungstate (NaW) is an effective antidiabetic agent for short and long-term treatments of both type 1 and type 2 diabetes models. In this study, we evaluated the effect of NaW on ROS production in bovine neutrophils incubated with platelet-activating factor (PAF) and in HK-2 cells induced by high glucose or H2O2. In addition, we evaluated the effect on iNOS expression in the type 1 diabetic rat model induced with streptozotocin (STZ). NaW inhibited ROS production in PAF-induced bovine neutrophils, and human tubular cells (HK-2) were incubated in high glucose or H2O2. In addition, NaW inhibited iNOS expression in glomeruli and tubular cells in the type 1 diabetic rat. This study demonstrates a new role for NaW as an active antioxidant and its potential use in treating DN.

Polymers

The development of fish oral vaccines is of great interest to the aquaculture industry due to the... more The development of fish oral vaccines is of great interest to the aquaculture industry due to the possibility of rapid vaccination of a large number of animals at reduced cost. In a previous study, we evaluated the effect of alginate-encapsulated Piscirickettsia salmonis antigens (AEPSA) incorporated in feed, effectively enhancing the immune response in Atlantic salmon (Salmo salar). In this study, we seek to characterize AEPSA produced by ionic gelation using an aerodynamically assisted jetting (AAJ) system, to optimize microencapsulation efficiency (EE%), to assess microparticle stability against environmental (pH, salinity and temperature) and gastrointestinal conditions, and to evaluate microparticle incorporation in fish feed pellets through micro-CT-scanning. The AAJ system was effective in obtaining small microparticles (d < 20 μm) with a high EE% (97.92%). Environmental conditions (pH, salinity and temperature) generated instability in the microparticles, triggering prote...

Frontiers in Immunology, 2021

An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for s... more An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1β and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only th...

Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosi... more Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosis in salmonids farms in Chile. It was recently reported that P. salmonis produces exotoxins that play a role in the pathogenesis. However a delivery system has not yet been identified. Outer membrane vesicles (OMVs) are 10-300 nm spherical-bilayer structures discharged from the surface of many Gram-negative bacteria, which are able to deliver toxins and virulence factors. Recently, we described the production of OMVs by P. salmonis in both, in normal growth in broth and inside the host cells after infection. However, pro-immflamatory cytokines expression induced by P. salmonis OMVs has not yet been characterized. Thus, the aim of this study was to investigate if OMVs purified from P. salmonis are able to generate an immflamatory immune response in fish macrophages. P. salmonis was grown in basal broth supplemented with Cysteine (3.18 mM) and ferric chloride (0.05 mM) at 18°C until early ...

Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the et... more Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the etiologic agent of Piscirickettsiosis, a systemic infection of multiple organs and tissues among as kidney, liver, spleen, brain, intestine, ovaries, and gills in several salmonids species such as Rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch), causing high mortality and serious economic losses for fish farming in the south of Chile. P. salmonis is able to infect, evade the immune response and replicate inside of several fish cell lines such as RTS-11, SHK-1 and CHSE-214. However, there are no reports concerning the infection and lifestyle of P. salmonis inside SHK-1 cell line. Thus, the aim of this study was to characterize, in a time-course, the infection and intracellular lifestyle of P. salmonis in SHK-1 fish cell line. SHK-1 cells were cultured at 20oC in 75 cm flask, in Leibovitz’s L-15 medium supplemented with 10% FBS. P. sa...

Microorganisms, 2020

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with... more Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and ses...

Scientific Reports, 2020

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-... more Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-economic impacts for Chilean salmonid aquaculture. The identification of major environmental reservoirs for P. salmonis has long been ignored. Most microbial life occurs in biofilms, with possible implications in disease outbreaks as pathogen seed banks. Herein, we report on an in vitro analysis of biofilm formation by P. salmonis Psal-103 (LF-89-like genotype) and Psal-104 (EM-90-like genotype), the aim of which was to gain new insights into the ecological role of biofilms using multiple approaches. The cytotoxic response of the salmon head kidney cell line to P. salmonis showed interisolate differences, depending on the source of the bacterial inoculum (biofilm or planktonic). Biofilm formation showed a variable-length lag-phase, which was associated with wider fluctuations in biofilm viability. Interisolate differences in the lag phase emerged regardless of the nutritional content of ...

Microorganisms, 2020

Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish ... more Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by...