Single-Nucleotide Polymorphism and Copy Number Variation of the Multidrug Resistance-1 Locus of Plasmodium vivax: Local and Global Patterns

Emerging resistance to chloroquine (CQ) poses a major challenge for Plasmodium vivax malaria control, and nucleotide substitutions and copy number variation in the P. vivax multidrug resistance 1 (pvmdr-1) locus, which encodes a digestive vacuole membrane transporter, may modulate this phenotype. We describe patterns of genetic variation in pvmdr-1 alleles from Acre and Amazonas in northwestern Brazil, and compare then with those reported in other malaria-endemic regions. The pvmdr-1 mutation Y976F, which is associated with CQ resistance in Southeast Asia and Oceania, remains rare in northwestern Brazil (1.8%) and its prevalence mirrors that of CO resistance worldwide. Gene amplification of pvmdr-1, which is associated with mefloquine resistance but increased susceptibility to CO, remains relatively rare in northwestern Brazil (0.9%) and globally (< 4%), but became common (> 10%) in Tak Province, Thailand, possibly because of drug-mediated selection. The global database we have assembled provides a baseline for further studies of genetic variation in pvmdr-1 and drug resistance in P. vivax malaria.

Early and late neurodegeneration and memory disruption after intracerebroventricular streptozotocin

Glucose metabolism and insulin signaling disruptions in the brain have been proposed as a likely etiology of Alzheimer's disease. The aim of the present study was to investigate the time course of cognitive impairments induced by intracerebroventricular injection of streptozotocin (STZ) in rats and correlate them with the ensuing neurodegenerative process. Early and late effects of STZ were evaluated by using the reference and working memory versions of the Morris' water maze task and the evaluation of neurodegenerative markers by immunoblotting and the Fluoro-jade C histochemistry. The results revealed different types of behavioral and neurodegenerative responses, with distinct time courses. We observed an early disruption on the working memory as early as 3 h after STZ injections, which was followed by degenerative processes in the hippocampus at 1 and 15 days after STZ injections. Memory disruption increases over time and culminates with significant changes in amyloid-beta peptide and hyperphosphorylated Tau protein levels in distinct brain structures. These findings add information on the Alzheimer's disease-like STZ animal model and on the mechanisms underlying neurodegenerative processes. (C) 2012 Elsevier Inc. All rights reserved.

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

Abstract Background Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. Results In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-β-glucosidase) in mycelium cells; and ags (an α-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. Conclusion Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.

Effects of intravenous furosemide on mucociliary transport and rheological properties of patients under mechanical ventilation

The use of intravenous (IV) furosemide is common practice in patients under mechanical ventilation (MV), but its effects on respiratory mucus are largely unknown. Furosemide can affect respiratory mucus either directly through inhibition of the NaK(Cl)2 co-transporter on the basolateral surface of airway epithelium or indirectly through increased diuresis and dehydration. We investigated the physical properties and transportability of respiratory mucus obtained from 26 patients under MV distributed in two groups, furosemide (n = 12) and control (n = 14). Mucus collection was done at 0, 1, 2, 3 and 4 hours. The rheological properties of mucus were studied with a microrheometer, and in vitro mucociliary transport (MCT) (frog palate), contact angle (CA) and cough clearance (CC) (simulated cough machine) were measured. After the administration of furosemide, MCT decreased by 17 ± 19%, 24 ± 11%, 18 ± 16% and 18 ± 13% at 1, 2, 3 and 4 hours respectively, P < 0.001 compared with control. In contrast, no significant changes were observed in the control group. The remaining parameters did not change significantly in either group. Our results support the hypothesis that IV furosemide might acutely impair MCT in patients under MV.

Genome-wide metabolic (re-) annotation of Kluyveromyces lactis

Background: Even before having its genome sequence published in 2004, Kluyveromyces lactis had long been considered a model organism for studies in genetics and physiology. Research on Kluyveromyces lactis is quite advanced and this yeast species is one of the few with which it is possible to perform formal genetic analysis. Nevertheless, until now, no complete metabolic functional annotation has been performed to the proteins encoded in the Kluyveromyces lactis genome. Results: In this work, a new metabolic genome-wide functional re-annotation of the proteins encoded in the Kluyveromyces lactis genome was performed, resulting in the annotation of 1759 genes with metabolic functions, and the development of a methodology supported by merlin (software developed in-house). The new annotation includes novelties, such as the assignment of transporter superfamily numbers to genes identified as transporter proteins. Thus, the genes annotated with metabolic functions could be exclusively enzymatic (1410 genes), transporter proteins encoding genes (301 genes) or have both metabolic activities (48 genes). The new annotation produced by this work largely surpassed the Kluyveromyces lactis currently available annotations. A comparison with KEGG’s annotation revealed a match with 844 (~90%) of the genes annotated by KEGG, while adding 850 new gene annotations. Moreover, there are 32 genes with annotations different from KEGG. Conclusions: The methodology developed throughout this work can be used to re-annotate any yeast or, with a little tweak of the reference organism, the proteins encoded in any sequenced genome. The new annotation provided by this study offers basic knowledge which might be useful for the scientific community working on this model yeast, because new functions have been identified for the so-called metabolic genes. Furthermore, it served as the basis for the reconstruction of a compartmentalized, genome-scale metabolic model of Kluyveromyces lactis, which is currently being finished.

A compound heterozygote SLC26A2 mutation resulting in robin sequence, mild limbs shortness, accelerated carpal ossification, and multiple epiphysial dysplasia in two Brazilian sisters. A new intermediate phenotype between diastrophic dysplasia and recessive multiple epiphyseal dysplasia

Mutations in solute carrier family 26 (sulfate transporter), member 2 (SLC26A2) gene result in a spectrum of autosomal recessive chondrodysplasias that range from the mildest recessive form of multiple epiphysial dysplasia (rMED) through the most common diastrophic dysplasia (DTD) to lethal atelosteogenesis type II and achondrogenesis IB. The clinical variability has been ascribed to quantitative effect of mutations of the sulfate transporter activity. Here we describe two Brazilian sisters, born to healthy and non consanguineous parents, with Robin sequence, mild shortening of upper and lower limbs, brachymetacarpalia/tarsalia, additional and accelerated carpal ossification, marked genu valgum, and multiple epiphysial dysplasia. This phenotype was intermediate between DTD and rMED, and both girls have a compound heterozygous mutations for the SLC26A2, a Finnish founder mutation (c.-26?+?2T>C), and R279W. This combination of mutations has been observed in individuals with different phenotypes, including DTD, DTD variant, and rMED. The distinct phenotype of our cases reinforces the hypothesis that other factors may be influencing the phenotype as previously suggested.

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats

Abstract Background: Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. Findings: By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. Conclusions: SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects.

Hepatocyte nuclear factors 1α/4α and forkhead box A2 regulate the solute carrier 2A2 (Slc2a2) gene expression in the liver and kidney of diabetic rats

AIMS: Solute carrier 2a2 (Slc2a2) gene codifies the glucose transporter GLUT2, a key protein for glucose flux in hepatocytes and renal epithelial cells of proximal tubule. In diabetes mellitus, hepatic and tubular glucose output has been related to Slc2a2/GLUT2 overexpression; and controlling the expression of this gene may be an important adjuvant way to improve glycemic homeostasis. Thus, the present study investigated transcriptional mechanisms involved in the diabetes-induced overexpression of the Slc2a2 gene. MAIN METHODS: Hepatocyte nuclear factors 1α and 4α (HNF-1α and HNF-4α), forkhead box A2 (FOXA2), sterol regulatory element binding protein-1c (SREBP-1c) and the CCAAT-enhancer-binding protein (C/EBPβ) mRNA expression (RT-PCR) and binding activity into the Slc2a2 promoter (electrophoretic mobility assay) were analyzed in the liver and kidney of diabetic and 6-day insulin-treated diabetic rats. KEY FINDINGS: Slc2a2/GLUT2 expression increased by more than 50% (P<0.001) in the liver and kidney of diabetic rats, and 6-day insulin treatment restores these values to those observed in non-diabetic animals. Similarly, the mRNA expression and the binding activity of HNF-1α, HNF-4α and FOXA2 increased by 50 to 100% (P<0.05 to P<0.001), also returning to values of non-diabetic rats after insulin treatment. Neither the Srebf1 and Cebpb mRNA expression, nor the SREBP-1c and C/EBP-β binding activity was altered in diabetic rats. SIGNIFICANCE: HNF-1α, HNF-4α and FOXA2 transcriptional factors are involved in diabetes-induced overexpression of Slc2a2 gene in the liver and kidney. These data point out that these transcriptional factors are important targets to control GLUT2 expression in these tissues, which can contribute to glycemic homeostasis in diabetes.

Adaptations of the aging animal to exercise: role of daily supplementation with melatonin

The pineal gland, through melatonin, seems to be of fundamental importance in determining the metabolic adaptations of adipose and muscle tissues to physical training. Evidence shows that pinealectomized animals fail to develop adaptive metabolic changes in response to aerobic exercise and therefore do not exhibit the same performance as control-trained animals. The known prominent reduction in melatonin synthesis in aging animals led us to investigate the metabolic adaptations to physical training in aged animals with and without daily melatonin replacement. Male Wistar rats were assigned to four groups: sedentary control (SC), trained control (TC), sedentary treated with melatonin (SM), and trained treated with melatonin (TM). Melatonin supplementation lasted 16 wk, and the animals were subjected to exercise during the last 8 wk of the experiment. After euthanasia, samples of liver, muscle, and adipose tissues were collected for analysis. Trained animals treated with melatonin presented better results in the following parameters: glucose tolerance, physical capacity, citrate synthase activity, hepatic and muscular glycogen content, body weight, protein expression of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and protein kinase activated by adenosine monophosphate (AMPK) in the liver, as well as the protein expression of the glucose transporter type 4 (GLUT4) and AMPK in the muscle. In conclusion, these results demonstrate that melatonin supplementation in aging animals is of great importance for the required metabolic adaptations induced by aerobic exercise. Adequate levels of circulating melatonin are, therefore, necessary to improve energetic metabolism efficiency, reducing body weight and increasing insulin sensitivity.

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats

Background Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. Findings By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. Conclusions SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects

Changes in renal glucose transporters in an animal model of metabolic syndrome

Considering the similarity between structural, hemodynamic, and functional changes of obesity-related renal disease and diabetic nephropathy, we hypothesized that renal glucose transporter changes occur in obesity as in diabetes. The aim of the work was to evaluate GLUT1 and GLUT2 in kidneys of an animal model of metabolic syndrome. Neonate spontaneously hypertensive rats (SHR), n=15/group, were treated with monosodium glutamate (5 mg/g) (MetS) for 9 days and compared with saline-treated Wistar-Kyoto (C) and SHR (H) rats. Lee index, systolic arterial pressure (SAP), glycemia, insulin resistance, triglycerides, and HDL cholesterol were evaluated at 3 and 6 months. Medullar GLUT1 and cortical GLUT2 were analyzed by Western blot. MetS vs. C and H rats had the highest Lee index (p<0.001) and insulin resistance (3-months C: 4.3±0.7, H: 3.9±0.9, MetS: 2.7±0.6; 6-months C: 4.2±0.6, H: 3.8±0.5, MetS: 2.4±0.6% • min−1, p<0.001), similar glycemia, and the lowest HDL-cholesterol at 6-months (p<0.001). In the MetS and H rats, SAP was higher vs. C at 3-months (p<0.001) and 6-months (C: 151±15, H: 190±11, MetS: 185±13 mm Hg, p<0.001) of age. GLUT1 was ̴ 13× lower (p<0.001) at 3-months, reestablishing its content at 6-months in MetS group, while GLUT2 was 2× higher (p<0.001) in this group at 6-months of age. Renal GLUT1 and GLUT2 are modulated in kidney of rats with metabolic syndrome, where obesity, insulin resistance and hypertension coexist, despite normoglycemia. Like in diabetes, cortical GLUT2 overexpression may contribute to the development of kidney disease

Renal GLUT1 reduction depends on angiotensin-converting enzyme inhibition in diabetic hypertensive rats

Aims: Angiotensin-converting enzyme (ACE) inhibitors are used in diabetic kidney disease to reduce systemic/intra-glomerular pressure. The objective of this study was to investigate whether reducing blood pressure (BP) could modulate renal glucose transporter expression, and urinary markers of diabetic nephropathy in diabetic hypertensive rats treated with ramipril or amlodipine. Main methods: Diabetes was induced in spontaneously-hypertensive rats (~210 g) by streptozotocin (50 mg/kg). Thirty days later, animals received ramipril 15 μg/kg/day (R, n =10), or amlodipine 10 mg/kg/day (A, n= 8,) or water (C, n = 10) by gavage. After 30-day treatment, body weight, glycaemia, urinary albumin and TGF-β1 (enzyme-linked immunosorbent assay) and BP (tail-cuff pressure method) were evaluated. Kidneys were removed for evaluation of renal cortex glucose transporters (Western blotting) and renal tissue ACE activity (fluorometric assay). Key findings: After treatments, body weight (p = 0.77) and glycaemia (p = 0.22) were similar among the groups. Systolic BP was similarly reduced (p < 0.001) in A and R vs. C (172.4 ± 3.2; 186.7 ± 3.7 and 202.2 ± 4.3 mm Hg; respectively). ACE activity (C: 0.903 ± 0.086; A: 0.654 ± 0.025, and R: 0.389 ± 0.057 mU/mg), albuminuria (C: 264.8 ± 15.4; A: 140.8 ± 13.5 and R: 102.8 ± 6.7 mg/24 h), and renal cortex GLUT1 content (C: 46.81 ± 4.54; A: 40.30 ± 5.39 and R: 26.89 ± 0.79 AU) decreased only in R (p < 0.001, p < 0.05 and p < 0.001; respectively). Significance:We concluded that the blockade of the renin–angiotensin systemwith ramipril reduced earlymarkers of diabetic nephropathy, a phenomenon that cannot be specifically related to decreased BP levels.

Aerobic exercise training induces metabolic benefits in rats with metabolic syndrome independent of dietary changes

OBJECTIVES: We evaluated the effects of aerobic exercise training without dietary changes on cardiovascular and metabolic variables and on the expression of glucose transporter Type 4 in rats with metabolic syndrome. METHODS: Twenty male spontaneously hypertensive rats received monosodium glutamate during the neonatal period. The animals were allocated to the following groups: MS (sedentary metabolic syndrome), MS-T (trained on a treadmill for 1 hour/day, 5 days/week for 10 weeks), H (sedentary spontaneously hypertensive rats) and H-T (trained spontaneously hypertensive rats). The Lee index, blood pressure (tail-cuff system), insulin sensitivity (insulin tolerance test) and functional capacity were evaluated before and after 10 weeks of training. Glucose transporter Type 4 expression was analyzed using Western blotting. The data were compared using analysis of variance (ANOVA) (p<0.05). RESULTS: At baseline, the MS rats exhibited lower insulin sensitivity and increased Lee index compared with the H rats. Training decreased the body weight and Lee index of the MS rats (MS-T vs. MS), but not of the H rats (H-T vs. H). There were no differences in food intake between the groups. At the end of the experiments, the systolic blood pressure was lower in the two trained groups than in their sedentary controls. Whole-body insulin sensitivity increased in the trained groups. Glucose transporter Type 4 content increased in the heart, white adipose tissue and gastrocnemius muscle of the trained groups relative to their respective untrained groups. CONCLUSION: In conclusion, the present study shows that an isolated aerobic exercise training intervention is an efficient means of improving several components of metabolic syndrome, that is, training reduces obesity and hypertension and increases insulin sensitivity

Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia

[EN] Chronic hypoxia is accompanied by changes in blood and skeletal muscle acid-base control. We hypothesized that the underlying mechanisms include altered protein expression of transport systems and the enzymes involved in lactate, HCO3- and H+ fluxes in skeletal muscle and erythrocytes. Immunoblotting was used to quantify densities of the transport systems and enzymes. Muscle and erythrocyte samples were obtained from eight Danish lowlanders at sea level and after 2 and 8 weeks at 4100 m (Bolivia). For comparison, samples were obtained from eight Bolivian natives. In muscle membranes there were no changes in fibre-type distribution, lactate dehydrogenase isoforms, Na+,K+-pump subunits or in the lactate-H+ co-transporters MCT1 and MCT4. The Na+-H+ exchanger protein NHE1 was elevated by 39 % in natives compared to lowlanders. The Na+-HCO3- co-transporter density in muscle was elevated by 47-69 % after 2 and 8 weeks at altitude. The membrane-bound carbonic anhydrase (CA) IV in muscle increased in the lowlanders by 39 %, whereas CA XIV decreased by 23-47 %. Levels of cytosolic CA II and III in muscle and CA I and II in erythrocytes were unchanged. The erythrocyte lactate-H+ co-transporter MCT1 increased by 230-405 % in lowlanders and was 324 % higher in natives. The erythrocyte inorganic anion exchanger (Cl--HCO3- exchanger AE1) was increased by 149-228 %. In conclusion, chronic hypoxia induces dramatic changes in erythrocyte proteins, but only moderate changes in muscle proteins involved in acid-base control. Together, these changes suggest a hypoxia-induced increase in the capacity for lactate, HCO3- and H+ fluxes from muscle to blood and from blood to erythrocytes.

PepT1 mRNA expression levels in sea bream (Sparus aurata) fed different plant protein sources

[EN] The expression and regulation of intestinal oligopeptide transporter (PepT)-1 when vegetable sources are used as a substitute for fish meal in the diet of marine fish has not yet been explored. In the present study, as part of our ongoing work on elucidating PepT1 gene expression in relation to different dietary treatments, we have now isolated and deposited in Genbank database (accession no. GU733710) a cDNA sequence representing the PepT1 in the sea bream (Sparus aurata). The ?de novo? prediction of the three-dimensional structure of PepT1 protein is presented. We also analyzed diet-induced changes in the expression of PepT1 mRNA via real-time RT-PCR using the standard curve method. Sea bream were fed for 140 days with one of the following four diet formulations (43% protein/21% lipid): a control fast growth-promoting diet (C), and three diets with the same formulation but in which 15% of the fish meal was substituted by protein concentrates either from lupine (LPC), chick pea (CPC), or green pea (PPC). Fish fed PPC had significantly (p < 0.05) lower levels of PepT1 transcripts in the proximal intestine than the controls, whereas PepT1 transcript levels in fish fed LPC or CPC were not significantly different from the controls. Although growth was similar between fish fed with different diets during the first 72 days of feeding, growth of the fish fed with PPC was reduced during the second part of the trial and was significantly (p < 0.05) lower than fish fed LPC and CPC diets by the end of the experiment. Correlation between these results and fish growth performances highlights that the intestinal PepT1 mRNA level may serve as a useful marker of the dietary protein quality and absorption efficiency.