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Copper is an essential micronutrient that functions as an enzymatic cofactor in a wide range of cellular processes. Although adequate Cu levels are essential for normal metabolism, excess Cu can be toxic to cells. Cellular responses to copper deficiency and overload involve changes in the expression of genes directly and indirectly involved in copper metabolism. However little is known on the effect of physiological copper concentration on gene expression changes. In the current study we aimed to establish whether the expression of genes encoding enzymes related to cholesterol (hmgcs1, hmgcr, fdft) and fatty acid biosynthesis and LDL receptor can be induced by an iso-physiological copper concentration. The iso-physiological copper concentration was determined as the bioavailable plasmatic copper in a healthy adult population. In doing so, two blood cell lines (Jurkat and THP-1) were exposed for 6 or 24 h to iso- or supraphysiological copper concentrations. Our results indicated that in cells exposed to an iso-physiological copper concentration the early induction of genes involved in lipid metabolism was not mediated by copper itself but by the modification of the cellular redox status. Thus our results contributed to understand the involvement of copper in the regulation of cholesterol metabolism under physiological conditions.

Celiac disease (CD) is an immune-mediated enteropathy involving genetic and environmental factors whose interaction might influence disease risk. The aim of this study was to determine the effects of milk-feeding practices and the HLA-DQ genotype on intestinal colonization of Bacteroides species in infants at risk of CD development. This study included 75 full-term newborns with at least one first-degree relative suffering from CD. Infants were classified according to milk-feeding practice (breast-feeding or formula feeding) and HLA-DQ genotype (high or low genetic risk). Stools were analyzed at 7 days, 1 month, and 4 months by PCR and denaturing gradient gel electrophoresis (DGGE). The Bacteroides species diversity index was higher in formula-fed infants than in breast-fed infants. Breast-fed infants showed a higher prevalence of Bacteroides uniformis at 1 and 4 months of age, while formula-fed infants had a higher prevalence of B. intestinalis at all sampling times, of B. caccae at 7 days and 4 months, and of B. plebeius at 4 months. Infants with high genetic risk showed a higher prevalence of B. vulgatus, while those with low genetic risk showed a higher prevalence of B. ovatus, B. plebeius, and B. uniformis. Among breast-fed infants, the prevalence of B. uniformis was higher in those with low genetic risk than in those with high genetic risk. Among formula-fed infants, the prevalence of B. ovatus and B. plebeius was increased in those with low genetic risk, while the prevalence of B. vulgatus was higher in those with high genetic risk. The results indicate that both the type of milk feeding and the HLA-DQ genotype influence the colonization process of Bacteroides species, and possibly the disease risk.

Background: Niemann-Pick type C disease (NPC) is a neurovisceral lipid storage disorder mainly characterized by unesterified cholesterol accumulation in lysosomal/late endosomal compartments, although there is also an important storage for several other kind of lipids. The main tissues affected by the disease are the liver and the cerebellum. Oxidative stress has been described in various NPC cells and tissues, such as liver and cerebellum. Although considerable alterations occur in the liver, the pathological mechanisms involved in hepatocyte damage and death have not been clearly defined. Here, we assessed hepatic tissue integrity, biochemical and oxidative stress parameters of wild-type control (Npc1(+/+); WT) and homozygous-mutant (Npc1(-/-); NPC) mice. In addition, the mRNA abundance of genes encoding proteins associated with oxidative stress, copper metabolism, fibrosis, inflammation and cholesterol metabolism were analyzed in livers and cerebella of WT and NPC mice., Methodology/principal Findings: We analyzed various oxidative stress parameters in the liver and hepatic and cerebellum gene expression in 7-week-old NPC1-deficient mice compared with control animals. We found signs of inflammation and fibrosis in NPC livers upon histological examination. These signs were correlated with increased levels of carbonylated proteins, diminished total glutathione content and significantly increased total copper levels in liver tissue. Finally, we analyzed liver and cerebellum gene expression patterns by qPCR and microarray assays. We found a correlation between fibrotic tissue and differential expression of hepatic as well as cerebellar genes associated with oxidative stress, fibrosis and inflammation in NPC mice., Conclusions/significance: In NPC mice, liver disease is characterized by an increase in fibrosis and in markers associated with oxidative stress. NPC is also correlated with altered gene expression, mainly of genes involved in oxidative stress and fibrosis. These findings correlate with similar parameters in cerebellum, as has been previously reported in the NPC mice model., (© 2011 Vázquez et al.)

As a result of copper essentiality for life, plants and most other organisms have developed a conserved and complex network of proteins to handling Cu in order to prevent its deficit and to avoid its potentially toxic effects. To better understand regulation of Cu homeostasis in plants, we use adult plant of Arabidopsis thaliana to provide an integrated view of how Cu status affects the expression of genes involved in cellular Cu homoeostasis. In doing so, we use real-time RT-PCR to compare shoot and roots transcriptional responses to Cu. We measure changes in the abundance of transcripts encoding transporters, chaperones and P-type ATPases and correlated those changes with variation of Cu content in both tissues. Our results indicated that in both tissues transcript levels of COPT2, 4, and ZIP2 transporters and CCH chaperone were significantly down-regulated comparing to controls plants in response to Cu excess. In contrast, Cu chaperones ATX1, CCS, COX17-1 including two putative mitochondrial chaperones (At3g08950; At1g02410) were up-regulated under similar conditions. Regarding P-type ATPases, a reduction of HMA1, PAA1, PAA2, and RAN1 transcript levels in shoot after Cu exposure was observed, while HMA5 transcripts increased exclusively in roots. In plants growing under Cu-deficient conditions, COPT2, ZIP2, HMA1, andPAA2, were significantly up-regulated in shoots. Thus, our results indicated a common transcriptional regulation pattern of transporters and chaperone components, in particular transcriptional changes of COPT2, ZIP2, and CCH showed an inverse relation with Cu content suggesting that these proteins are required to avoid excess and deficit of Cu., (2010 Elsevier Inc. All rights reserved.)

Coeliac disease (CD) development involves genetic (HLA-DQ2/DQ8) and environmental factors. Herein, the influence of the HLA-DQ genotype on the gut colonization process of breast-fed children was determined. A cohort of 20 newborns, with at least one first-degree relative with CD, were classified according to their HLA-DQ genotype into high, intermediate and low genetic risk groups, showing 24-28%, 7-8% and less than 1% probability to develop CD, respectively. Faecal microbiota was analysed at 7 days, 1 and 4 months of children's age by fluorescence in situ hybridization. When considering all data, Gram-negative bacteria and Bacteroides-Prevotella group proportions were higher (P<0.05) in the high than in the intermediate and low genetic risk groups. E. coli, Streptococcus-Lactococcus, E. rectale-C. coccoides, sulphate-reducing bacteria, C. lituseburense and C. histolyticum group proportions were also significantly higher (P<0.05) in the high than in the low genetic risk group. Correlations between these bacterial groups and the genetic risk were also detected (P<0.05). In addition, the number and type of CD relative seemed to influence (P<0.050) these bacterial proportions in children at CD risk. At 4 months of age, similar relationships were established between the high genetic risk to develop CD and the proportions of Streptococcus-Lactococcus (P<0.05), E. rectale-C. coccoides (P<0.05), C. lituseburense (P<0.05), C. histolyticum (P<0.05), Bacteroides-Prevotella (P<0.10) groups and total Gram-negative bacteria (P<0.05). The results suggest a relationship between HLA-DQ genes and the gut microbial colonization process that could lead to a change in the way this disorder is investigated.

Sterol receptor element-binding protein-1c (SREBP-1c) and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) mRNA expression was assessed in liver as signaling mechanisms associated with steatosis in obese patients. Liver SREBP-1c and PPAR-alpha mRNA (RT-PCR), fatty acid synthase (FAS) and carnitine palmitoyltransferase-1a (CPT-1a) mRNA (real-time RT-PCR), and n-3 long-chain polyunsaturated fatty acid (LCPUFA)(GLC) contents, plasma adiponectin levels (RIA), and insulin resistance (IR) evolution (HOMA) were evaluated in 11 obese patients who underwent subtotal gastrectomy with gastro-jejunal anastomosis in Roux-en-Y and 8 non-obese subjects who underwent laparoscopic cholecystectomy (controls). Liver SREBP-1c and FAS mRNA levels were 33% and 70% higher than control values (P<0.05), respectively, whereas those of PPAR-alpha and CPT-1a were 16% and 65% lower (P<0.05), respectively, with a significant 62% enhancement in the SREBP-1c/PPAR-alpha ratio. Liver n-3 LCPUFA levels were 53% lower in obese patients who also showed IR and hipoadiponectinemia over controls (P<0.05). IR negatively correlated with both the hepatic content of n-3 LCPUFA (r=-0.55; P<0.01) and the plasma levels of adiponectin (r=-0.62; P<0.005). Liver SREBP-1c/PPAR-alpha ratio and n-3 LCPUFA showed a negative correlation (r=-0.48; P<0.02) and positive associations with either HOMA (r=0.75; P<0.0001) or serum insulin levels (r=0.69; P<0.001). In conclusion, liver up-regulation of SREBP-1c and down-regulation of PPAR-alpha occur in obese patients, with enhancement in the SREBP-1c/PPAR-alpha ratio associated with n-3 LCPUFA depletion and IR, a condition that may favor lipogenesis over FA oxidation thereby leading to steatosis.

Background: Questionnaires are feasible instruments to assess physical activity (PA) in large samples. The aim of the current study was to evaluate the reliability and validity of the PAQ-A questionnaire in Spanish adolescents using the measurement of PA by accelerometer as criterion., Methods: In a sample of 82 adolescents, aged 12 to 17 years, 1-week PAQ-A test-retest was administered. Reliability was analyzed by the Intraclass Correlation Coefficient (ICC) and the internal consistency by the Cronbach's alpha Coefficient. Two hundred thirty-two adolescents, aged 13-17 years, completed the PAQ-A and wore the ActiGraph GT1M accelerometer during 7-days. The PAQ-A was compared against total PA and moderate to vigorous PA (MVPA) obtained by the accelerometer., Results: Test-retest reliability showed ICC = 0.71 for the final score of PAQ-A. Internal consistency was alpha = 0.65 in the first self-report, alpha = 0.67 in the retest in 82 adolescents sample, and alpha = 0.74 in the 232 adolescents sample. The PAQ-A was moderately correlated with total PA (rho = 0.39) and MVPA (rho= 0.34) assessed by the accelerometer. The PAQ-A obtained significantly moderate correlations in boys but not in girls against the accelerometer., Conclusions: The PAQ-A questionnaire shows an adequate reliability and a reasonable validity for assessing PA in Spanish adolescents.

A subtractive hybridization approach was used to identify genes that are expressed at the beginning of gastrulation. We used tester DNA complimentary to RNA (cDNA) prepared from stages 6-7 embryos (gastrula) and excess driver cDNA from stages 2-4 embryos (syncytial blastoderm) to generate a gastrula-subtracted cDNA library. A reverse Northern blot procedure used to analyze 105 subtracted clones showed that 65% had a level of expression at least 2.5-fold higher in stages 6-7 versus stages 2-4 embryos. We determined the nucleotide sequence of these clones and identified 49 individual sequences, including 33 previously uncharacterized genes. We verified the level of expression of 24 genes during Drosophila melanogaster embryogenesis using a semiquantitative polymerase chain reaction (PCR) approach. As expected, all of the selected clones showed their highest level of expression after stages 2-4 of embryogenesis, including several that displayed peaks of expression during gastrulation. Three genes that were expressed at their highest levels in stages 6-7 were further analyzed by 5'-rapid amplification of cDNA ends (RACE) analysis, Northern blot assays and in situ hybridization. Our results indicate that these genes exhibited temporal and spatially restricted patterns of expression in developing embryos, and moreover, their transcripts were detected in cells that undergo morphological changes during the gastrulation stage. Characterizing the role of these genes will be important to increase our understanding of the molecular mechanisms that regulate cellular activities during D. melanogaster gastrulation.

Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.

An 80-year-old man with a 7 year history of a slowly enlarging, asymptomatic scrotal nodule is presented. He had a negative history for sexually transmitted disease, trauma to the area, radiotherapy and chemical or arsenic exposure. The lesion was excised with a margin of 0.8 cm of normal skin. Examination of the specimen revealed a basal cell carcinoma.

Cocaine base or white coca paste was smoked heavily by 188 patients who came to four hospitals of Lima, Peru. The length of hospitalization varied from two days to six months. All patients were admitted because coca paste smoking had become a serious problem for their health or for social adjustment. Coca paste was smoked mixed with tobacco or marihuana. The main symptoms were anxiety mingled with euphoria and a rapidly developing compulsion to continue smoking. Frequently the patients developed irritability, illusions, hallucinations. When the use of the drug was heavy and continued for many hours or several days, the patients developed successively four stages of psychological reaction: euphoria, dysphoria, hallucinosis, and acute paranoid psychosis. The main symptoms and signs of all of these phases are discussed.