Juan Pablo Pontigo

Nuclear accumulation of fructose 1,6-bisphosphatase is impaired in diabetic rat liver

Using a streptozotocin‐induced type 1 diabetic rat model, we analyzed and separated the effects of hyperglycemia and hyperinsulinemia over the in vivo expression and subcellular localization of hepatic fructose 1,6‐bisphosphatase (FBPase) in the multicellular context of the liver. Our data showed that FBPase subcellular localization was modulated by the nutritional state in normal but not in diabetic rats. By contrast, the liver zonation was not affected in any condition. In healthy starved rats, FBPase was localized in the cytoplasm of hepatocytes, whereas in healthy re‐fed rats it was concentrated in the nucleus and the cell periphery. Interestingly, despite the hyperglycemia, FBPase was unable to accumulate in the nucleus in hepatocytes from streptozotocin‐induced diabetic rats, suggesting that insulin is a critical in vivo modulator. This idea was confirmed by exogenous insulin supplementation to diabetic rats, where insulin was able to induce the rapid accumulation of FBPase within the hepatocyte nucleus. Besides, hepatic FBPase was found phosphorylated only in the cytoplasm, suggesting that the phosphorylation state is involved in the nuclear translocation. In conclusion, insulin and not hyperglycemia plays a crucial role in the nuclear accumulation of FBPase in vivo and may be an important regulatory mechanism that could account for the increased endogenous glucose production of liver of diabetic rodents. J. Cell. Biochem. 113: 848–856, 2012.

Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis

To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally.

Identification, characterization and modulation of ferritin-H in the sub-Antarctic Notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis

&NA; Ferritin is a major iron storage protein essential not only in the infectious process, but also in any circumstance generating oxidative stress. In this study, the cDNA coding sequence of ferritin‐H was obtained from the sub‐Antarctic Notothenioid fish Eleginops maclovinus through transcriptomic analysis of the head kidney. This sequence contained a 534 bp open reading frame that coded for a 177 amino acid protein with a molecular weight of 20,786.2 Da and a theoretical pI of 5.56. The protein displayed a region of iron putative response elements in the 5′UTR, two putative ferritin iron‐binding region signatures, and seven characteristic amino acids with ferroxidase functions. Phylogenetic analysis related this sequence to ferritin‐H sequences of other Antarctic Notothenioid fish, sharing 96.61% similarity. Constitutive gene expression analysis in different organs revealed increased ferritin‐H gene expression in the gills, spleen, muscle, and liver. After infection with two bacterial strains of Piscirickettsia salmonis (LF‐89 and Austral‐005), ferritin‐H was differentially expressed depending on bacterial strain and tissue. This study provides relevant information towards understanding the iron metabolism of a sub‐Antarctic Notothenioid fish. HighlightsFerritin is a major iron storage protein essential in the infectious process.Phylogenetic related this sequence to ferritin‐H sequences of other Antarctic Notothenioid fish, sharing 96.61% homology.Ferritin‐H was differentially expressed depending on bacterial strain type of P. salmonis and tissue.

Voltage-dependent BK and Hv1 channels expressed in non-excitable tissues: New therapeutics opportunities as targets in human diseases

Voltage-gated ion channels are the molecular determinants of cellular excitability. This group of ion channels is one of the most important pharmacological targets in excitable tissues such as nervous system, cardiac and skeletal muscle. Moreover, voltage-gated ion channels are expressed in non-excitable cells, where they mediate key cellular functions through intracellular biochemical mechanisms rather than rapid electrical signaling. This review aims at illustrating the pharmacological impact of these ion channels, highlighting in particular the structural details and physiological functions of two of them - the high conductance voltage- and Ca(2+)-gated K(+) (BK) channels and voltage-gated proton (Hv1) channels- in non-excitable cells. BK channels have been implicated in a variety of physiological processes ranging from regulation of smooth muscle tone to modulation of hormone and neurotransmitter release. Interestingly, BK channels are also involved in modulating K(+) transport in the mammalian kidney and colon epithelium with a potential role in the hyperkalemic phenotype observed in patients with familial hyperkalemic hypertension type 2, and in the pathophysiology of hypertension. In addition, BK channels are responsible for resting and stimulated Ca(2+)-activated K(+) secretion in the distal colon. Hv1 channels have been detected in many cell types, including macrophages, blood cells, lung epithelia, skeletal muscle and microglia. These channels have a central role in the phagocytic system. In macrophages, Hv1 channels participate in the generation of reactive oxygen species in the respiratory burst during the process of phagocytosis.

Nutritional Immunity Triggers the Modulation of Iron Metabolism Genes in the Sub-Antarctic Notothenioid Eleginops maclovinus in Response to Piscirickettsia salmonis

Iron deprivation is a nutritional immunity mechanism through which fish can limit the amount of iron available to invading bacteria. The aim of this study was to evaluate the modulation of iron metabolism genes in the liver and brain of sub-Antarctic notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis. The specimens were inoculated with two P. salmonis strains: LF-89 (ATCC® VR-1361™) and Austral-005 (antibiotic resistant). Hepatic and brain samples were collected at intervals over a period of 35 days. Gene expression (by RT-qPCR) of proteins involved in iron storage, transport, and binding were statistically modulated in infected fish when compared with control counterparts. Specifically, the expression profiles of the transferrin and hemopexin genes in the liver, as well as the expression profiles of ferritin-M, ferritin-L, and transferrin in the brain, were similar for both experimental groups. Nevertheless, the remaining genes such as ferritin-H, ceruloplasmin, hepcidin, and haptoglobin presented tissue-specific expression profiles that varied in relation to the injected bacterial strain and sampling time-point. These results suggest that nutritional immunity could be an important immune defense mechanism for E. maclovinus against P. salmonis injection. This study provides relevant information for understanding iron metabolism of a sub-Antarctic notothenioid fish.

Temperature modulates the immunological response of the sub-antarctic notothenioid fish Eleginops maclovinus injected with Piscirickettsia salmonis

Abstract Eleginops maclovinus is a eurythermic fish that under natural conditions lives in environments with temperatures ranging from 4 to 18 °C and can be usually captured near salmon farming areas. The aim of this study was to evaluate the effect of temperature over the innate and adaptive immune response of E. maclovinus challenged with Piscirickettsia salmonis following different treatments: C— (control injection with culture medium at 12 °C), C+ (bacterial injection at 12 °C), 18 °C c/A + B (injection with culture medium in acclimation at 18 °C), 18 °C c/A + B (bacterial injection in acclimation at 18 °C), 18 °C s/A + M (injection with culture medium without acclimation at 18 °C) and 18 °C s/A + B (bacterial injection without acclimation at 18 °C). Each injection had 100 &mgr;L of culture medium or with 100 &mgr;L at a concentration 1 × 108 of live bacteria, sampling six fish per group at 4, 8, 12, 16 and 20 days post‐injection (dpi). Expression of the mRNA related with the innate immune response gene (TLR1, TLR5, TLR8, NLRC3, NLRC5, MyD88 and IL‐1&bgr;) as well as the adaptive immune response gene (MHCI, MHCII, IgMs and IgD) were measured in spleen and head kidney. Gene expression profiles were treatment‐type and time dependent. Levels of Immunoglobulin M (IgM) increased in challenged groups with P. salmonis from day 8–20 post challenge, which suggest activation of B cells IgM + through P. salmonis epitope detection. Additionally, a rise in temperature from 12 °C (C+) to 18 °C (with/without acclimation) also resulted in antibody increment detected in serum with significant differences between “18 °C c/A + B” and “18 °C s/A + B” groups. This is the first study that evaluates the effect of temperature changes and mRNA expression related with immune system gene over time on E. maclovinus, a native wild life fish that cohabits in the salmon farming environment. HighlightsThe increase in temperature affects the gene transcription related with innate and adaptive immune response.The increase in temperature (with and without acclimatation) increased the amount of antibodies detected in serum.The temperature is modulating the immune response in Eleginops maclovinus.

Immunological response of the Sub-Antarctic Notothenioid fish Eleginops maclovinus injected with two strains of Piscirickettsia salmonis

ABSTRACT Eleginops maclovinus is an endemic fish to Chile that lives in proximity to salmonid culture centers, feeding off of uneaten pellet and salmonid feces. Occurring in the natural environment, this interaction between native and farmed fish could result in the horizontal transmission of pathogens affecting the aquaculture industry. The aim of this study was to evaluate the innate and adaptive immune responses of E. maclovinus challenged with P. salmonis. Treatment injections (in duplicate) were as follows: control (100 &mgr;L of culture medium), wild type LF‐89 strain (100 &mgr;L, 1 × 108 live bacteria), and antibiotic resistant strain Austral‐005 (100 &mgr;L, 1 × 108 live bacteria). The fish were sampled at various time‐points during the 35‐day experimental period. The gene expression of TLRs (1, 5, and 8), NLRCs (3 and 5), C3, IL‐1&bgr;, MHCII, and IgMs were significantly modulated during the experimental period in both the spleen and gut (excepting TLR1 and TLR8 spleen expressions), with tissue‐specific expression profiles and punctual differences between the injected strains. Anti‐P. salmonis antibodies increased in E. maclovinus serum from day 14–28 for the LF‐89 strain and from day 14–35 for the Austral‐005 strain. These results suggest temporal activation of the innate and adaptive immune responses in E. maclovinus tissues when injected by distinct P. salmonis strains. The Austral‐005 strain did not always cause the greatest increases/decreases in the number of transcripts, so the magnitude of the observed immune response (mRNA) may not be related to antibiotic resistance. This is the first immunological study to relate a pathogen widely studied in salmonids with a native fish. HighlightsEleginops maclovinus is living in proximity to salmonid culture centers feeding off of uneaten pellet and salmonid feces.The aim of this study was to evaluate the innate and adaptive immune responses of E. maclovinus challenged with P. salmonis.The results suggest activation of the immune response in E. maclovinus injected by distinct P. salmonis strains.This is the first immunological study to relate a pathogen widely studied in salmonids with a native fish.