Publications by authors named "Carolina Salvador"

20 Publications

  • Page 1 of 1

Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation.

FEBS Open Bio 2021 Feb 10. Epub 2021 Feb 10.

Departamento de Fisiologia, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Mexico.

G protein-activated inward-rectifying potassium (K ) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a noncanonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte meiotic progression. The chimeric proteins enhanced green fluorescent protein (EGFP)-GIRK5-WT and the EGFP-GIRK5K13AR14A double mutant, were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K channel evolved to control Xenopus oocyte maturation.
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http://dx.doi.org/10.1002/2211-5463.13113DOI Listing
February 2021

Metabolic acidosis and hyperkalemia differentially regulate cation HCN3 channel in the rat nephron.

J Mol Histol 2020 Dec 18;51(6):701-716. Epub 2020 Oct 18.

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), 04510, Mexico, Mexico.

The kidney controls body fluids, electrolyte and acid-base balance. Previously, we demonstrated that hyperpolarization-activated and cyclic nucleotide-gated (HCN) cation channels participate in ammonium excretion in the rat kidney. Since acid-base balance is closely linked to potassium metabolism, in the present work we aim to determine the effect of chronic metabolic acidosis (CMA) and hyperkalemia (HK) on protein abundance and localization of HCN3 in the rat kidney. CMA increased HCN3 protein level only in the outer medulla (2.74 ± 0.31) according to immunoblot analysis. However, immunofluorescence assays showed that HCN3 augmented in cortical proximal tubules (1.45 ± 0.11) and medullary thick ascending limb of Henle's loop (4.48 ± 0.45) from the inner stripe of outer medulla. HCN3 was detected in brush border membranes (BBM) and mitochondria of the proximal tubule by immunogold electron and confocal microscopy in control conditions. Acidosis did not alter HCN3 levels in BBM and mitochondria but augmented them in lysosomes. HCN3 was also immuno-detected in mitoautophagosomes. In the distal nephron, HCN3 was expressed in principal and intercalated cells from cortical to medullary collecting ducts. CMA did not change HCN3 abundance in these nephron segments. In contrast, HK doubled HCN3 level in cortical collecting ducts and favored its basolateral localization in principal cells from the inner medullary collecting ducts. These findings further support HCN channels contribution to renal acid-base and potassium balance.
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http://dx.doi.org/10.1007/s10735-020-09916-2DOI Listing
December 2020

"Funny" channels in cardiac mitochondria modulate membrane potential and oxygen consumption.

Biochem Biophys Res Commun 2020 04 14;524(4):1030-1036. Epub 2020 Feb 14.

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City, 04510, Mexico. Electronic address:

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by a family of four genes (HCN1-4). All isoforms are expressed in the heart, HCN4 being the most abundant in the sinoatrial node (SAN). HCN channels are responsible for the "funny" current (I) associated with the generation and autonomic control of the diastolic depolarization phase of cardiac action potential. In this work we performed a proteomic analysis of HCN4 transfected in HEK293 cells. Most of the identified proteins in the HCN4 network belonged to mitochondria. The subcellular localization of HCN channels was predicted in plasma membrane, mitochondria and nucleus. Experimentally, HCN2 (full-length, truncated), HCN3 (full-length, truncated) and HCN4 (truncated) were detected in rat heart mitochondria by immunoblotting. I sensitive to ZD7288, was recorded by patch-clamp in mitoplasts from cardiomyocytes. Mitochondrial membrane potential (ΔΨm) assessment in H9c2 cells revealed that ZD7288 induced almost 50% higher hyperpolarization respect to control at 30 min. Furthermore, ZD7288 reduced oxygen consumption attributed to ATP synthesis in H9c2 cells. In conclusion, we identify for the first time functional HCN channels in mammalian cardiac mitochondria and demonstrate their impact on ΔΨm and respiration.
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http://dx.doi.org/10.1016/j.bbrc.2020.02.033DOI Listing
April 2020

Patient perspectives on primary care and oncology care coordination in the context of multiple chronic conditions: A systematic review.

Res Social Adm Pharm 2020 Aug 26;16(8):1003-1016. Epub 2019 Nov 26.

Auburn University, Harrison School of Pharmacy, Health Outcomes Research and Policy, 4306 Walker Building, Auburn University, AL, 36849, USA; China Medical University Hospital, Department of Medical Research, No.2 Yude Road, North District, Taichung City, 40447, Taiwan. Electronic address:

Background: Patients' views on the optimal model for care coordination between primary care providers (PCPs) and oncologists in the context of cancer and multiple chronic conditions (MCC) are unclear. Thus, the purpose of this systematic review is to evaluate the perceptions of patients with both cancer and MCC regarding their care coordination needs.

Methods: Following PRISMA guidelines, the literature was systematically searched through PubMed, CINAHL, and PsycINFO for articles pertaining to patients' perspectives, experiences, and needs regarding care coordination between PCPs and oncologists during the cancer care continuum, in the context of patients with cancer and MCC. English-language articles were included if they met the following criteria: 1) published between 2008 and 2018; 2) peer-reviewed study; 3) patients aged 18 years or older diagnosed with any type or stage of cancer; 4) patients have one or more chronic comorbid condition; 5) inclusion of patient perceptions, experiences, or needs related to care coordination between PCPs and oncologists; and 6) ability to extract results. Data extraction was performed with a standardized form, and themes were developed through qualitative synthesis. A grounded theory approach was used to qualitatively evaluate data extracted from articles and create a framework for providers to consider when developing patient-centered care coordination strategies for these complex patients. Risk of bias within each study was assessed independently by two authors using the Mixed Methods Appraisal Tool.

Results: A total of 22 articles were retained, representing the perspectives of 8,114 patients with cancer and MCC. Studies were heterogeneous in the patients' respective phases of cancer care and study design. From qualitative synthesis, four themes emerged regarding patients' needs for cancer care coordination and were included as constructs to develop the Patient-centered Care Coordination among Patients with Multiple Chronic Conditions and Cancer (PCP-MC) framework. Constructs included: 1) Communication; 2) Defining provider care roles; 3) Information access; and 4) Individualized patient care. Care navigators served as a communication bridge between providers and patients.

Conclusions: Findings highlight the importance that patients with both cancer and MCC place on communication with and between providers, efficient access to understandable care information, defined provider care roles, and care tailored to their individual needs and circumstances. Providers and policymakers may consider the developed PCP-MC framework when designing, implementing, and evaluating patient-centered care coordination strategies for patients with both cancer and MCC.
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http://dx.doi.org/10.1016/j.sapharm.2019.11.014DOI Listing
August 2020

Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels.

Int J Mol Sci 2019 Oct 9;20(20). Epub 2019 Oct 9.

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510, Mexico.

Hyperpolarization-activated cationic HCN channels comprise four members (HCN1-4) that control dendritic integration, synaptic transmission and action potential firing. In the kidney, HCN1, HCN2 and HCN3 are differentially expressed and contribute to the transport of sodium, potassium (K) and ammonium into the nephrons. HCN3 is regulated by K diets in the kidney. In this work we performed a proteomic analysis of HCN3 expressed in human embryonic kidney cells (HEK293 cells). More than 50% of the interacting proteins belonged to mitochondria. Therefore, we explored the presence of HCN channels in kidney mitochondria. By immunoblotting and immunogold electron microscopy HCN3 protein expression was found in rat kidney mitochondria; it was also confirmed in human kidney. Patch-clamp recordings of renal mitochondria and mitochondria from HEK293 cells overexpressing HCN1, HCN2 and HCN3 channels, stained with MitoTracker Green FM, indicated that only HCN3 could produce inwardly K currents that were inhibited by ZD7288, a specific blocker of HCN channels. Furthermore, ZD7288 caused inhibition of the oxygen consumption coupled to ATP synthesis and hyperpolarization of the inner mitochondrial membrane. In conclusion, we show for the first time that pacemaker HCN channels contribute to K transport in mitochondria facilitating the activity of the respiratory chain and ATP synthesis by controlling the inner mitochondrial membrane potential.
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http://dx.doi.org/10.3390/ijms20204995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834191PMC
October 2019

Mind-Body Interventions for Individuals With Heart Failure: A Systematic Review of Randomized Trials.

J Card Fail 2018 Mar 20;24(3):186-201. Epub 2017 Sep 20.

School of Nursing, University of Alabama at Birmingham, Birmingham, Alabama.

Background: The effects of mind-body interventions (MBIs) (eg, Tai Chi, yoga, meditation) for individuals with heart failure (HF) have not been systematically evaluated.

Methods And Results: We performed a systematic review of randomized controlled trials (RCTs) examining the effects of MBIs in HF. We extracted participant characteristics, MBI procedure, outcomes assessed, and main results of English-language RCTs before October 2016. We identified 24 RCTs (n = 1314 participants) of 9 MBI types: Tai Chi (n = 7), yoga (n = 4), relaxation (n = 4), meditation (n = 2), acupuncture (n = 2), biofeedback (n = 2), stress management (n = 1), Pilates (n = 1), and reflexology (n = 1). Most (n = 22, 95.8%) reported small-to-moderate improvements in quality of life (14/14 studies), exercise capacity (8/9 studies), depression (5/5 studies), anxiety and fatigue (4/4 studies), blood pressure (3/5 studies), heart rate (5/6 studies), heart rate variability (7/9 studies), and B-type natriuretic peptide (3/4 studies). Studies ranged from 4 minutes to 26 weeks and group sizes ranged from 8 to 65 patients per study arm.

Conclusions: Although wide variability exists in the types and delivery, RCTs of MBIs have demonstrated small-to-moderate positive effects on HF patients' objective and subjective outcomes. Future research should examine the mechanisms by which different MBIs exert their effects.
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http://dx.doi.org/10.1016/j.cardfail.2017.09.008DOI Listing
March 2018

Mutations in ATP6V1B1 and ATP6V0A4 genes cause recessive distal renal tubular acidosis in Mexican families.

Mol Genet Genomic Med 2016 May 14;4(3):303-11. Epub 2016 Feb 14.

Département de Génetique Hôpital Européen Georges Pompidou Paris France.

Background: Autosomal recessive distal renal tubular acidosis (dRTA) is a rare disease characterized by a hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia, nephrocalcinosis, and conserved glomerular filtration rate. In some cases, neurosensorial deafness is associated. dRTA is developed during the first months of life and the main manifestations are failure to thrive, vomiting, dehydration, and anorexia.

Methods: Nine unrelated families were studied: seven children, a teenager, and an adult with dRTA. Hearing was preserved in four children. Coding regions of the genes responsible for recessive dRTA were analysed by Sanger sequencing.

Results: Molecular defects were found in the genes ATP6V1B1 and ATP6V0A4. We identified three homozygous variants in ATP6V1B: a frameshift mutation (p.Ile386Hisfs*56), a nucleotide substitution in exon 10 (p.Pro346Arg), and a new splicing mutation in intron 5. Three patients were homozygous for one novel (p.Arg743Trp) and one known (p.Asp411Tyr) missense mutations in the ATP6V0A4 gene. Three patients were compound heterozygous: one proband displayed two novel mutations, the frameshift mutation p.Val52Metfs*25, and a large deletion of exons 18-21; two probands showed the missense mutation p.Asp411Tyr and as a second mutation, p.Arg194Ter and c.1691+2dup, respectively.

Conclusion: ATP6V0A4 and ATP6V1B1 genes were involved in recessive dRTA of Mexican families. All ATP6V1B1 mutations detected were homozygous and all patients developed sensorineural hearing loss (SNHL) early in infancy. ATP6V0A4 mutations were found in one infant and three children without SNHL, and in one teenager and one adult with SNHL confirming the phenotypic variability in this trait. The mutation p.Asp411Tyr detected in four Mexican families was due to a founder effect. Screening of these mutations could provide a rapid and valuable tool for diagnosis of dRTA in this population.
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http://dx.doi.org/10.1002/mgg3.205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867564PMC
May 2016

Influences of diet and the gut microbiome on epigenetic modulation in cancer and other diseases.

Clin Epigenetics 2015 16;7:112. Epub 2015 Oct 16.

Department of Biology, University of Alabama at Birmingham, 175 Campbell Hall, 1300 University Boulevard, Birmingham, AL 35294-1170 USA ; Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL USA ; Comprehensive Center for Healthy Aging, University of Alabama at Birmingham, Birmingham, AL USA ; Nutrition Obesity Research Center, University of Alabama at Birmingham, Birmingham, AL USA ; Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, AL USA.

Epigenetic modulation of gene activity occurs in response to non-genetic factors such as body weight status, physical activity, dietary factors, and environmental toxins. In addition, each of these factors is thought to affect and be affected by the gut microbiome. A primary mechanism that links these various factors together in mediating control of gene expression is the production of metabolites that serve as critical cofactors and allosteric regulators of epigenetic processes. Here, we review the involvement of the gut microbiota and its interactions with dietary factors, many of which have known cellular bioactivity, focusing on particular epigenetic processes affected and the influence they have on human health and disease, particularly cancer and response to treatment. Advances in DNA sequencing have expanded the capacity for studying the microbiome. Combining this with rapidly improving techniques to measure the metabolome provides opportunities to understand complex relationships that may underlie the development and progression of cancer as well as treatment-related sequelae. Given broad reaching and fundamental biology, both at the cellular and organismal levels, we propose that interactive research programs, which utilize a wide range of mutually informative experimental model systems-each one optimally suited for answering particular questions-provide the best path forward for breaking ground on new knowledge and ultimately understanding the epigenetic significance of the gut microbiome and its response to dietary factors in cancer prevention and therapy.
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http://dx.doi.org/10.1186/s13148-015-0144-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609101PMC
October 2015

Cancer-Related Fatigue, Version 2.2015.

J Natl Compr Canc Netw 2015 Aug;13(8):1012-39

Cancer-related fatigue is defined as a distressing, persistent, subjective sense of physical, emotional, and/or cognitive tiredness or exhaustion related to cancer or cancer treatment that is not proportional to recent activity and interferes with usual functioning. It is one of the most common side effects in patients with cancer. Fatigue has been shown to be a consequence of active treatment, but it may also persist into posttreatment periods. Furthermore, difficulties in end-of-life care can be compounded by fatigue. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Cancer-Related Fatigue provide guidance on screening for fatigue and recommendations for interventions based on the stage of treatment. Interventions may include education and counseling, general strategies for the management of fatigue, and specific nonpharmacologic and pharmacologic interventions. Fatigue is a frequently underreported complication in patients with cancer and, when reported, is responsible for reduced quality of life. Therefore, routine screening to identify fatigue is an important component in improving the quality of life for patients living with cancer.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499710PMC
http://dx.doi.org/10.6004/jnccn.2015.0122DOI Listing
August 2015

Differential expression of the Kv1 voltage-gated potassium channel family in the rat nephron.

J Mol Histol 2014 Oct 20;45(5):583-97. Epub 2014 Jun 20.

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510, México, DF, Mexico.

Several potassium (K(+)) channels contribute to maintaining the resting membrane potential of renal epithelial cells. Apart from buffering the cell membrane potential and cell volume, K(+) channels allow sodium reabsorption in the proximal tubule (PT), K(+) recycling and K(+) reabsorption in the thick ascending limb (TAL) and K(+) secretion and K(+) reabsorption in the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct. Previously, we identified Kv.1.1, Kv1.3 and Kv1.6 channels in collecting ducts of the rat inner medulla. We also detected intracellular Kv1.3 channel in the acid secretory intercalated cells, which is trafficked to the apical membrane in response to dietary K(+) to function as a secretory K(+) channel. In this work we sought to characterize the expression of all members of the Kv1 family in the rat nephron. mRNA and protein expression were detected for all Kv1 channels. Immunoblots identified differential expression of each Kv1 in the cortex, outer and inner medulla. Immunofluorescence labeling detected Kv1.5 in Bowman´s capsule and endothelial cells and Kv1.7 in podocytes, endothelial cells and macula densa in glomeruli; Kv1.4, Kv1.5 and Kv1.7 in PT; Kv1.2, Kv1.4 and Kv1.6 in TAL; Kv1.1, Kv1.4 and Kv1.6 in DCT and CNT and Kv1.3 in DCT, and all the Kv1 family in the cortical and medullary collecting ducts. Recently, some hereditary renal syndromes have been attributed to mutations in K(+) channels. Our results expand the repertoire of K(+) channels that contribute to K(+) homeostasis to include the Kv1 family.
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http://dx.doi.org/10.1007/s10735-014-9581-4DOI Listing
October 2014

The polarization of the G-protein activated potassium channel GIRK5 to the vegetal pole of Xenopus laevis oocytes is driven by a di-leucine motif.

PLoS One 2013 22;8(5):e64096. Epub 2013 May 22.

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, México.

The G protein-coupled inwardly-rectifying potassium channels (known as GIRK or Kir3) form functional heterotetramers gated by G-βγ subunits. GIRK channels participate in heart rate modulation and neuronal postsynaptic inhibition in mammals. In Xenopus laevis oocytes, GIRK5 is a functional homomultimer. Previously, we found that phosphorylation of a tyrosine (Y16) at its N-terminus downregulates the surface expression of GIRK5. In this work, we elucidated the subcellular localization and trafficking of GIRK5 in oocytes. Several EGFP-GIRK5 chimeras were produced and an ECFP construct was used to identify the endoplasmic reticulum (ER). Whereas GIRK5-WT was retained in the ER at the animal pole, the phospho-null GIRK5-Y16A was localized to the vegetal pole. Interestingly, a construct with an N-terminal Δ25 deletion produced an even distribution of the channel in the whole oocyte. Through an alanine-scan, we identified an acidic cluster/di-leucine sorting-signal recognition motif between E17 and I22. We quantified the effect of each amino acid residue within this di-leucine motif in determining the distribution of GIRK5 to the animal and vegetal poles. We found that Y16 and I22 contributed to functional expression and were dominant in the polarization of GIRK5. We thus conclude that the N-terminal acidic di-leucine motif of GIRK5 determines its retention and polarized trafficking within Xl oocytes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064096PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661522PMC
April 2014

The hyperpolarization-activated cyclic nucleotide-gated HCN2 channel transports ammonium in the distal nephron.

Kidney Int 2011 Oct 27;80(8):832-40. Epub 2011 Jul 27.

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, México DF, México.

Recent studies have identified Rhesus proteins as important molecules for ammonia transport in acid-secreting intercalated cells in the distal nephron. Here, we provide evidence for an additional molecule that can mediate NH3/NH4 excretion, the subtype 2 of the hyperpolarization-activated cyclic nucleotide-gated channel family (HCN2), in collecting ducts in rat renal cortex and medulla. Chronic metabolic acidosis in rats did not alter HCN2 protein expression but downregulated the relative abundance of HCN2 mRNA. Its cDNA was identical to the homolog from the brain and the protein was post-translationally modified by N-type glycosylation. Electrophysiological recordings in Xenopus oocytes injected with HCN2 cRNA found that potassium was transported better than ammonium, each of which was transported significantly better than sodium, criteria that are compatible with a role for HCN2 in ammonium transport. In microperfused rat outer medullary collecting duct segments, the initial rate of acidification, upon exposure to a basolateral ammonium chloride pulse, was higher in intercalated than in principal cells. A specific inhibitor of HCN2 (ZD7288) decreased acidification only in intercalated cells from control rats. In rats with chronic metabolic acidosis, the rate of acidification doubled in both intercalated and principal cells; however, ZD7288 had no significant inhibitory effect. Thus, HCN2 is a basolateral ammonium transport pathway of intercalated cells and may contribute to the renal regulation of body pH under basal conditions.
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http://dx.doi.org/10.1038/ki.2011.230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752342PMC
October 2011

Potassium secretion by voltage-gated potassium channel Kv1.3 in the rat kidney.

Am J Physiol Renal Physiol 2010 Jul 28;299(1):F255-64. Epub 2010 Apr 28.

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, México.

The fine regulation of Na(+) and K(+) transport takes place in the cortical distal nephron. It is well established that K(+) secretion occurs through apical K(+) channels: the ROMK and the Ca(2+)- and voltage-dependent maxi-K. Previously, we identified the voltage-gated Kv1.3 channel in the inner medulla of the rat kidney (Escobar LI, Martínez-Téllez JC, Salas M, Castilla SA, Carrisoza R, Tapia D, Vázquez M, Bargas J, Bolívar JJ. Am J Physiol Cell Physiol 286: C965-C974, 2004). To examine the role of Kv1.3 in the renal regulation of K(+) homeostasis, we characterized the effect of dietary K(+) on the molecular and functional expression of this channel. We performed real-time-PCR and immunoblot assays in kidneys from rats fed a control (CK; 1.2% wt/wt) or high-K(+) (HK; 10% wt/wt) diet for 5-15 days. Kv1.3 mRNA and protein expression did not change with HK in the whole kidney. However, dietary K(+) loading provoked a change in the cellular distribution of Kv1.3 from the cytoplasm to apical membranes. Immunolocalization of Kv1.3 detected the channel exclusively in the intercalated cells. We investigated whether Kv1.3 mediated K(+) transport in microperfused cortical collecting ducts (CCDs). The HK diet led to an increase in net K(+) transport from 7.4 +/- 1.1 (CK) to 11.4 +/- 1.0 (HK) pmol x min(-1.) mm(-1). Luminal margatoxin, a specific blocker of Kv1.3, decreased net K(+) secretion in HK CCDs to 6.0 +/- 1.6 pmol x min(-1.) mm(-1). Our data provide the first evidence that Kv1.3 channels participate in K(+) secretion and that apical membrane localization of Kv1.3 is enhanced in the intercalated cells by dietary K(+) loading.
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http://dx.doi.org/10.1152/ajprenal.00697.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904170PMC
July 2010

Peptide sr11a from Conus spurius is a novel peptide blocker for Kv1 potassium channels.

Peptides 2010 Jul 18;31(7):1287-91. Epub 2010 Apr 18.

Laboratorio de Neurofarmacología Marina, Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, Mexico.

More than a hundred conotoxins are known today and from them, only seven conopeptides have been identified to target voltage-gated potassium channels (Kv). Conotoxin sr11a belongs to the I(2)-superfamily which is characterized by four disulfide bridges and provokes muscle stiffness when injected intracranially in mice. The aim of this work was to test the biological activity of sr11a on recombinant voltage-gated Kv1 potassium channels expressed in Xenopus laevis oocytes. Peptide sr11a was purified by high-performance liquid chromatography from the venom of the vermivorous Conus spurius. We found that peptide sr11a inhibits the delayed rectifiers Kv1.2 and Kv1.6 but had not effect on the slowly inactivating Kv1.3 channel. The functional dyad composed of a basic Lys and a hydrophobic amino acid residue is a crucial structural element, regarding the binding properties and blocking activities of more than a hundred K(+) channel toxins. Peptide sr11a does not contain Lys residues and then, it lacks the functional dyad. Molecular modeling of peptide sr11a reveals the presence of exposed basic residues of Arg and suggests that Arg17 and Arg29 are important on its biological activity.
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http://dx.doi.org/10.1016/j.peptides.2010.04.007DOI Listing
July 2010

Expression and immunolocalization of ERG1 potassium channels in the rat kidney.

Histochem Cell Biol 2010 Feb 17;133(2):189-99. Epub 2009 Nov 17.

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, DF, Mexico.

Potassium (K(+)) channels participate in K(+) secretion, K(+) recycling, and cell volume regulation and help to maintain the resting potential in mammalian kidneys. Previously, we identified a set of voltage-gated K(+) channels (Kv1) in the inner medullary collecting duct of the rat kidney. In the present work, we identified the voltage-gated K(+) channel ether-à-go-go-related gene (ERG) in the rat kidney. mRNAs of ERG1a and its N-terminal splice-variant ERG1b were detected. Immunoblots of the cortex and medulla revealed two molecular mass proteins of 135 and 80 kDa, consistent in size with the nonglycosylated ERG1a and ERG1b isoforms, respectively. However, bands of 155 and 95 kDa, corresponding to mature glycosylated ERG1a and ERG1b, respectively, were also observed. In our immunohistochemical experiments, we could not differentiate the ERG1 isoforms because we used an antibody against a carboxy-terminal epitope. ERG1 was differentially localized in specific nephron segments: its localization was intracellular in the proximal tubule and medullary collecting ducts and in the apical membranes in the distal convoluted and connecting tubules. ERG1 was also abundant in glomerular arterioles and renal vessels. In summary, ERG1 displays a heterogeneous distribution in the rat kidney.
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http://dx.doi.org/10.1007/s00418-009-0658-1DOI Listing
February 2010

Effect of yeast-derived beta-glucan in conjunction with bevacizumab for the treatment of human lung adenocarcinoma in subcutaneous and orthotopic xenograft models.

J Immunother 2009 Sep;32(7):703-12

Tumor Immunobiology Program of the James Graham Brown Cancer Center, Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40202, USA.

Human lung cancer is the leading cause of cancer death worldwide. Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), in combination with chemotherapy showed significant therapeutic efficacy in human lung cancer patients. However, increased adverse effects limit its clinical utilization. Previous studies demonstrated that polysaccharide beta-glucan significantly augments antitumor monoclonal antibody-mediated efficacy via stimulation of the innate effector neutrophil complement receptor 3. Here, we explored combined beta-glucan with bevacizumab therapy for human lung cancer using murine xenograft models. To that end, human lung adenocarcinomas were screened for membrane-bound VEGF expression. Both subcutaneous and orthotopic lung cancer xenograft models were used to evaluate the combination therapy. We found that PC14PE6 adenocarcinoma cells express membrane-bound VEGF both in vitro and in vivo. Bevacizumab bound to surface VEGF on PC14PE6 cells and activated complement. In the subcutaneous PC14PE6 tumor model, beta-glucan plus bevacizumab showed augmented efficacy in terms of tumor progression and long-term survival compared with bevacizumab-treated alone. These effects were accompanied with massive complement deposition and neutrophil infiltration within tumors. However, this effect was not observed in surface-bound VEGF-negative human lung tumors. Therapeutic efficacy of beta-glucan with bevacizumab was further demonstrated in an orthotopic lung cancer model. Thus, our data suggest that beta-glucan enhances bevacizumab-mediated efficacy and may provide therapeutic benefits for lung cancers with membrane-bound VEGF expression.
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http://dx.doi.org/10.1097/CJI.0b013e3181ad3fcfDOI Listing
September 2009

Yeast-derived beta-glucan augments the therapeutic efficacy mediated by anti-vascular endothelial growth factor monoclonal antibody in human carcinoma xenograft models.

Clin Cancer Res 2008 Feb;14(4):1239-47

Tumor Immunobiology Program of the James Graham Brown Cancer Center and Division of Hematology/Oncology, Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40202, USA.

Purpose: Bevacizumab is a recombinant IgG1 humanized monoclonal antibody against vascular endothelial growth factor (VEGF). Its proposed mechanism of action is independent of immune effector functions. Many human carcinomas not only secrete VEGF but also express membrane-bound VEGF. In addition, VEGF receptors are expressed on tumor cells. It is hypothesized that bevacizumab could bind membrane-bound VEGF or VEGF-VEGF receptor complexes on tumors, thereby initiating potential immunologic consequences. We previously showed that yeast-derived beta-glucan functions with antitumor antibodies that activate complement to recruit complement receptor 3-expressing leukocytes capable of mediating complement receptor 3-dependent cellular cytotoxicity of tumors opsonized with iC3b. In the current study, the therapeutic efficacy mediated by combining bevacizumab with yeast-derived beta-glucan was studied in human carcinoma xenograft models.

Experimental Design: Human tumor cell lines were screened for membrane-bound VEGF expression both in vitro and in vivo. Complement activation mediated by bevacizumab was examined. Tumor cell lines positive or negative for membrane-bound VEGF expression were implanted in severe combined immunodeficient mice to establish xenograft models. Tumor-bearing mice were treated with different regimens. Tumor regression and long-term survival were recorded.

Results: Human ovarian carcinoma SKOV-3 cells expressed membrane-bound VEGF both in vitro and in vivo. Bevacizumab was bound to membrane-bound VEGF, activated complement, and synergized with beta-glucan to elicit cellular cytotoxicity in vitro. In vivo study showed that beta-glucan could significantly augment the therapeutic efficacy mediated by bevacizumab.

Conclusions: Yeast-derived beta-glucan can synergize with anti-VEGF monoclonal antibody bevacizumab for the treatment of cancer with membrane-bound VEGF expression.
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http://dx.doi.org/10.1158/1078-0432.CCR-07-1669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2394864PMC
February 2008

A phase I study of thalidomide, capecitabine and temozolomide in advanced cancer.

Cancer Biol Ther 2007 Jun 5;6(6):840-5. Epub 2007 Mar 5.

Division of Hematology and Medical Oncology, University of Louisville, J.G. Brown Cancer Center, Louisville, Kentucky 40202, USA.

Aim: To develop a novel oral antiangiogenic and immunomodulatory chemotherapy regimen against advanced cancers.

Methods: Patients were enrolled in cohorts of three or six in a standard phase I design. Thalidomide 100 mg was kept stable for all cohorts. If well tolerated after one week, capecitabine and temozolomide (TCT) fixed-dose were given daily without rest and escalated one at a time until dose limiting toxicity (DLT) occurred. If no DLT occurred within 28 days (1 cycle), the subsequent group was enrolled into the next dose level.

Results: Twenty-three adult patients with objective documentation of progressive metastatic cancer after a median of two therapies (range 0-4) received TCT in this study. Based on DLT within the first 28 days, the MTD of daily TCT therapy was thalidomide 100 mg, capecitabine 2000 mg and temozolomide 100 mg. With long-term treatment, majority of the patients required dose interruptions due to fatigue, hand-foot syndrome, and neutropenia. A dose level consisting of daily thalidomide and three-weeks-on one-week-off capecitabine and temozolomide was implemented and administered to six patients without DLT. Out of 23 subjects, four (17%) achieved a partial response (renal, gastric, prostate 2), and four patients (17%) had stable disease (renal, colon, pancreas 2), adding to a clinical benefit of 34%.

Conclusions: TCT is an effective palliative oral chemo-immunotherapy for patients with advanced cancer. The recommended dose for phase II trial is thalidomide 100 mg daily without a break, capecitabine 2000 mg and temozolomide 100 mg daily three-weeks on and one-week off.
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http://dx.doi.org/10.4161/cbt.6.6.4099DOI Listing
June 2007

Basal activity of GIRK5 isoforms.

Life Sci 2003 Feb;72(13):1509-18

Departamento de Fisiologi;a, Facultad de Medicina, UNAM, Apartado Postal 70-250, D.F., 04510, Mexico.

G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results.
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http://dx.doi.org/10.1016/s0024-3205(02)02447-5DOI Listing
February 2003