Association between genetic variations at 8q24 and prostate cancer risk in Mexican Men.

BACKGROUND: Variants of 8q24 locus have been associated with prostate cancer (PCa) susceptibility. This study aims to analyze the genetic basis of PCa susceptibility in Mexican men by analyzing SNPs in the 8q24 locus for the first time. METHODS: A case-control study was performed in 875 men recruited from the Mexican Social Security Institute, 326 patients with PCa, and 549 non-PCa patients (88 with benign prostatic hyperplasia BPH and 461 healthy controls). The 8q24 locus SNPs: rs16901979, rs16983267, rs1447295, and rs7837328 were genotyped by allelic discrimination assays using TaqMan probes. Statistical analysis was performed using Epi Info statistical 7.0 and SNPstats softwares. RESULTS: All genotype frequencies were in Hardy-Weinberg Equilibrium. No differences were observed in genotype distribution between PCa and non-PCa patients for rs6983267. Under different inheritance models, the rs16901979, rs1447295, and rs7837328 SNPs were associated with PCa (OR = 2.8, 1.8, and 1.72, respectively; Pc < 0.001) when comparing PCa patients against controls. This association remains between PCa and BPH patients under different models (OR = 8.5, 2.2, and 1.9, respectively; Pc < 0.001). There were no significant differences in allele and genotype distribution among BPH patients and controls. The combined effect of the alleles CGAA for the SNPs rs16901979, rs6983267, rs1447295, and rs7837328 showed significant differences between PCa patients and controls (OR = 2.9, 95% CI = 1.48-5.83, Pc = 0.008). Four 8q24 variants were not associated with D'Amico score, age at diagnosis, and bone metastases. CONCLUSIONS: Our study provides the first confirmation that variants rs16901979, rs1447295, and 7837328 at 8q24 locus are associated with PCa susceptibility in Mexican men.

Punica granatum L.-derived omega-5 nanoemulsion improves hepatic steatosis in mice fed a high fat diet by increasing fatty acid utilization in hepatocytes.

Pomegranate seed oil (PSO) is mainly composed of punicic acid (PA), a polyunsaturated fatty acid also known as omega-5 (ω-5), a potent antioxidant associated with a variety of metabolic and cellular beneficial effects. However, the potential benefits of a nanoemulsified version of ω-5 (PSOn) have not been evaluated in a pathological liver condition. Here, we examined whether PSOn had beneficial effects on C57BL/6N mice fed a high-fat diet (HFD), specifically on hepatic steatosis. We observed that PSOn supplementation decreased body weight and body fat mass in control mice, whereas glucose intolerance, insulin resistance, energy expenditure, and hepatic steatosis were improved in both control mice and in mice fed a HFD. Interestingly, PSOn increased fatty acid oxidation in primary hepatocytes and antioxidant gene expression. Altogether, our data indicate that PSOn effectively reduces some of the HFD-derived metabolic syndrome indicators by means of an increase in fatty acid oxidation within hepatocytes.

A dietary intervention with functional foods reduces metabolic endotoxaemia and attenuates biochemical abnormalities by modifying faecal microbiota in people with type 2 diabetes.

AIM: To study the effects of a functional food-based dietary intervention on faecal microbiota and biochemical parameters in patients with type 2 diabetes (T2D). MATERIALS AND METHODS: This placebo-controlled, randomized, double-blind study included 81 patients with T2D divided into two 3-month treatment groups: one following a reduced-energy diet with a dietary portfolio (DP) comprising high-fibre, polyphenol-rich and vegetable-protein functional foods; the other taking a placebo (P). The primary outcome was the effect of the DP on faecal microbiota. Secondary endpoints were biochemical parameters, lipopolysaccharide, branched-chain amino acids, trimethylamine N-oxide, glycosylated haemoglobin (HbA1c) and free fatty acids (FFAs). RESULTS: Patients with T2D exhibited intestinal dysbiosis characterized by an increase in Prevotella copri. Dietary intervention with functional foods significantly modified faecal microbiota compared with P by increasing alpha diversity and modifying the abundance of specific bacteria, independently of antidiabetic drugs. There was a decrease in P. copri and increases in Faecalibacterium prausnitzii and Akkermansia muciniphila, two bacterial species known to have anti-inflammatory effects. The DP group also exhibited significant reductions in areas under the curve for glucose, total and LDL cholesterol, FFAs, HbA1c (P< 0.05), triglycerides and CRP, and an increase in antioxidant activity (P< 0.01) vs. the P group. CONCLUSION: Long-term adherence to a high-fibre, polyphenol-enriched and vegetable-protein-based diet provides benefits for the composition of faecal microbiota, and may offer potential therapies for improvement of glycaemic control, dyslipidaemia and inflammation.

Amino acid profiles of young adults differ by sex, body mass index and insulin resistance.

BACKGROUND AND AIMS: An increase in plasma branched-chain amino acids is associated with a higher risk of developing type 2 diabetes and cardiovascular diseases. However, little is known about the basal plasma amino acid concentrations in young adults. Our aim was to determine the plasma amino acid profiles of young adults and to evaluate how these profiles were modified by sex, body mass index (BMI) and insulin resistance (IR). METHODS AND RESULTS: We performed a transversal study with 608 Mexican young adults aged 19.9 ± 2.4 years who were applicants to the Universidad Autónoma de San Luis Potosí. The subjects underwent a physical examination and provided a clinical history and a blood sample for biochemical, hormonal and amino acid analyses. The women had higher levels of arginine, aspartate and serine and lower levels of α-aminoadipic acid, cysteine, isoleucine, leucine, methionine, proline, tryptophan, tyrosine, urea and valine than the men. The obese subjects had higher levels of alanine, aspartate, cysteine, ornithine, phenylalanine, proline and tyrosine and lower levels of glycine, ornithine and serine than the normal weight subjects. Subjects with IR (defined as HOMA > 2.5) had higher levels of arginine, alanine, aspartate, isoleucine, leucine, phenylalanine, proline, tyrosine, taurine and valine than the subjects without IR. Furthermore, we identified two main groups in the subjects with obesity and/or IR; one group was composed of amino acids that positively correlated with the clinical, biochemical and hormonal parameters, whereas the second group exhibited negative correlations. CONCLUSION: This study demonstrates that young adults with obesity or IR have altered amino acid profiles characterized by an increase in alanine, aspartate, proline and tyrosine and a decrease in glycine.

Fasting and postprandial regulation of the intracellular localization of adiponectin and of adipokines secretion by dietary fat in rats.

BACKGROUND/OBJECTIVE: Dietary fat sources modulate fasting serum concentration of adipokines, particularly adiponectin. However, previous studies utilized obese animals in which adipose tissue function is severely altered. Thus, the present study aimed to assess the postprandial regulation of adipokine secretion in nonobese rats that consumed high-fat diet (HFD) composed of different types of fat for a short time. METHODS: The rats were fed a control diet or a HFD containing coconut, safflower or soybean oil (rich in saturated fatty acid, monounsaturated fatty acid or polyunsaturated fatty acid, respectively) for 21 days. The serum concentrations of adiponectin, leptin, retinol, retinol-binding protein-4 (RBP-4), visfatin and resistin were determined at fasting and after refeeding. Adiponectin multimerization and intracellular localization, as well as the expression of endoplasmic reticulum (ER) chaperones and transcriptional regulators, were evaluated in epididymal white adipose tissue. RESULTS: In HFD-fed rats, serum adiponectin was significantly decreased 30 min after refeeding. With coconut oil, all three multimeric forms were reduced; with safflower oil, only the high-molecular-weight (HMW) and medium-molecular-weight (MMW) forms were decreased; and with soybean oil, only the HMW form was diminished. These reductions were due not to modifications in mRNA abundance or adiponectin multimerization but rather to an increment in intracellular localization at the ER and plasma membrane. Thus, when rats consumed a HFD, the type of dietary fat differentially affected the abundance of endoplasmic reticulum resident protein 44 kDa (ERp44), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-γ (PPARγ) mRNAs, all of which are involved in the post-translational processing of adiponectin required for its secretion.Leptin, RBP-4, resistin and visfatin serum concentrations did not change during fasting, whereas modest alterations were observed after refeeding. CONCLUSIONS: The short-term consumption of a HFD affected adiponectin localization in adipose tissue, thereby decreasing its secretion to a different magnitude depending on the dietary fat source. Evaluating the fasting serum concentration of adipokines was not sufficient to identify alterations in their secretion, whereas postprandial values provided additional information as dynamic indicators.

Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection-fluorescence in situ hybridisation.

The localisation and quantification of constitutive alkali-labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD-FISH) and alkaline single-cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD-FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility.

Soy protein isoflavones differentially regulate liver X receptor isoforms to modulate lipid metabolism and cholesterol transport in the liver and intestine in mice.

AIMS/HYPOTHESIS: Liver X receptor (LXR)α regulates the genes involved in cholesterol, fatty acid and glucose metabolism. Soy protein (SP) consumption reduces the hepatic accumulation of cholesterol and triacylglycerol, and improves insulin sensitivity. However, it is not known whether these effects are mediated via LXRα. We therefore investigated whether the consumption of SP regulates metabolic changes in cholesterol metabolism and insulin sensitivity via LXRα. METHODS: Wild-type (WT) and Lxrα(-/-) (Lxrα, also known as Nr1h3) mice were fed an SP diet with or without cholesterol for 28 days. The expression of LXRα target genes was measured in liver and intestine, as were hepatic lipid content and faecal bile acid concentration. Oral glucose and insulin tolerance tests were also performed. Hepatocytes were used to study the effect of isoflavones on LXR activity. RESULTS: The livers of WT and Lxrα(-/-) mice fed an SP high-cholesterol diet showed less steatosis than those fed casein. The SP diet increased the expression of the ATP-binding cassette (ABC) sub-family genes Abca1, Abcg5 and Abcg8 in the liver and intestine, as well as increasing total faecal bile acid excretion and insulin sensitivity in WT mice compared with mice fed a casein diet. However, these effects of SP were not observed in Lxrα(-/-) mice. The SP isoflavone, genistein, repressed the activation of LXRα target genes by T0901317, whereas it stimulated the activation of LXRß target genes. The AMP-activated protein kinase inhibitor, compound C, had the opposite effects to those of genistein. CONCLUSIONS/INTERPRETATION: Our results suggest that SP isoflavones stimulate the phosphorylation of LXRα or LXRß, resulting in different biological effects for each LXR isoform.

[Comparative study of burnout in Intensive Care and Emergency Care nursing staff]. / Estudio comparativo del burnout en personal de enfermería de Cuidados Intensivos y Urgencias.

OBJECTIVE: To assess and compare the burnout level between Intensive Care Unit and Emergency Unit, and study its association with the sociodemographic and work characteristics of the professionals surveyed. DESCRIPTION: Cross-sectional, descriptive study. Emplacement. Intensive Care Unit of the university hospital Morales Meseguer, Murcia-Spain. STUDIED SAMPLE: 97 nursing professionals: 55 professionals belong to the Emergency Department, and 42 professionals belong to the Intensive Care Department. METHOD: Two evaluation tools were used: a sociodemographic and work survey, and the Maslach Burnout Inventory, 1986. Quantitative variables expressed as mean +/- SD compared with the Student's T test and qualitative variables compared with the chi2 test. STATISTICAL ANALYSIS: SPSS 12.0(c). RESULTS: The comparative analysis of the burnout dimensions shows that emotional exhaustion level is significantly higher in the intensive care service than in the emergency one (25.45 +/- 11.15 vs 22.09 +/- 10.99) p < 0.05. The rest of burnout dimensions do not show significant differences between both departments. The masculine gender obtains a higher score in the depersonalization dimension of burnout (10.12 +/- 5.38) than female one (6.7 +/- 5.21) p < 0.01. There is greater vulnerability to emotional exhaustion among the professional group with more than 15 years of work experience (F = 3.592; p = 0.031). CONCLUSIONS: The burnout levels are moderate to high among the nursing professionals studied. A total of 5.15% of the sample studied achieves a high score in the three dimensions of the burnout syndrome. The intensive care professionals are the most vulnerable to suffering high levels of emotional exhaustion, and the masculine gender is more susceptible to depersonalization attitudes.

Estudio comparativo del burnout en personal de enfermería de Cuidados Intensivos y Urgencias / Comparative study of burnout in Intensive Care and Emergency Care nursing staff

Objetivo. Estimar y comparar el nivel de burnout existente en los Servicios de Cuidados Intensivos y Urgencias, y estudiar su asociación con las características sociodemográficas y laborales de los profesionales encuestados. Diseño. Estudio descriptivo transversal. Emplazamiento. Servicios de Urgencias y Cuidados Intensivos del Hospital Morales Meseguer (Murcia). Muestra. Noventa y siete profesionales de enfermería, 55 pertenecientes al Servicio de Urgencias y 42 al Servicio de Cuidados Intensivos. Método. Se emplearon dos instrumentos de evaluación: una encuesta de variables sociodemográficas y laborales, y el cuestionario Maslach Burnout Inventory, de 1986. Las variables cuantitativas se expresan como media ± desviación estándar comparadas con t de Student y las cualitativas se comparan con Chi2. El análisis de datos se realizó mediante el programa informático SPSS 12.0(C). Resultados. El análisis comparativo de las dimensiones que componen el burnout demuestra que los niveles de agotamiento emocional son significativamente mayores en el Servicio de Cuidados Intensivos que en el de Urgencias (25,45 ± 11,15 frente a 22,09 ± 10,99) p < 0,05. El resto de dimensiones que componen el síndrome no ha demostrado diferencias significativas entre ambos servicios. El género masculino obtiene una mayor puntuación en la dimensión de despersonalización (10,12 ± 5,38) que el género femenino (6,7 ± 5,21) p < 0,01. Existe una mayor vulnerabilidad al agotamiento emocional en el grupo de profesionales que llevan más de 15 años trabajando (F = 3,592; p = 0,031). Conclusiones. Los niveles encontrados de burnout resultaron ser moderados-altos. El 5,15% de la muestra total estudiada puntúa alto en las tres dimensiones del síndrome, los profesionales de Cuidados Intensivos son los más vulnerables a padecer elevados niveles de agotamiento emocional y el género masculino es el más propenso a las actitudes de despersonalización

Objective. To assess and compare the burnout level between Intensive Care Unit and Emergency Unit, and study its association with the sociodemographic and work characteristics of the professionals surveyed. Description. Cross-sectional, descriptive study. Emplacement. Intensive Care Unit of the university hospital Morales Meseguer, Murcia-Spain. Studied sample. 97 nursing professionals: 55 professionals belong to the Emergency Department, and 42 professionals belong to the Intensive Care Department. Method. Two evaluation tools were used: a sociodemographic and work survey, and the Maslach Burnout Inventory, 1986. Quantitative variables expressed as mean ± SD compared with the Student's T test and qualitative variables compared with the chi2 test. Statistical analysis: SPSS 12.0(C). Results. The comparative analysis of the burnout dimensions shows that emotional exhaustion level is significantly higher in the intensive care service than in the emergency one (25.45 ± 11.15 vs 22.09 ± 10.99) p < 0.05. The rest of burnout dimensions do not show significant differences between both departments. The masculine gender obtains a higher score in the depersonalization dimension of burnout (10.12 ± 5.38) than female one (6.7 ± 5.21) p < 0.01. There is greater vulnerability to emotional exhaustion among the professional group with more than 15 years of work experience (F = 3.592; p = 0.031). Conclusions. The burnout levels are moderate to high among the nursing professionals studied. A total of 5.15% of the sample studied achieves a high score in the three dimensions of the burnout syndrome. The intensive care professionals are the most vulnerable to suffering high levels of emotional exhaustion, and the masculine gender is more susceptible to depersonalization attitudes

Biochemical deficiency of pyridoxine does not affect interleukin-2 production of lymphocytes from patients with Sjögren's syndrome.

BACKGROUND: There is evidence that pyridoxine deficiency may alter the immune response. It is not known whether a deficiency of this vitamin is evident in subjects with primary Sjögren's syndrome (SS). OBJECTIVE: We studied whether subjects with primary SS showed a biochemical deficiency of pyridoxine, and if it is associated with abnormal production of interleukin-2 from lymphocytes stimulated in vitro with phytohemagglutinin (PHA). DESIGN: Two studies were conducted, (i) biochemical and nutritional assessments were performed in a cross-over study in subjects with primary SS, who were supplemented with 25 mg/day of pyridoxine or placebo for 3 months. After 1 month washout, they were supplemented for 3 months with placebo, (ii) patients with SS and matched controls received pyridoxine or placebo for 45 days, and a blood sample was obtained to study IL-2 production and expression in T-lymphocytes stimulated with PHA. RESULTS: Subjects with primary SS showed limited dietary intake of pyridoxine and biochemical deficiency of this vitamin assessed through the activation coefficient of the erythrocyte aspartate aminotransferase. The biochemical deficiency did not affect production nor mRNA expression of IL-2 from T-lymphocytes stimulated in vitro with PHA compared with the control group. Supplementation of subjects with primary SS with 25 mg/day with pyridoxine for 45 days did not produce any significant change as compared to those patients supplemented with placebo. CONCLUSIONS: Subjects with primary SS showed biochemical deficiency of pyridoxine, possibly due to limited intake of this vitamin which was corrected by supplementation with pyridoxine. However, IL-2 production and mRNA expression from stimulated lymphocytes were unaffected by supplementation, probably because the deficiency was not severe enough to affect the immune system. SPONSORSHIP: This work was supported by the National Council of Science and Technology (CONACYT), Mexico, grant no. 212226-5-0902PM.

Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid aminotransferase.

Branched chain amino-acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU x mg(-1) protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41-kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43-kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41-kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.

[Homocysteine metabolism and risk of cardiovascular diseases: importance of the nutritional status on folic acid, vitamins B6 and B12]. / Metabolismo de la homocisteína y riesgo de enfermedades cardiovasculares: importancia del estado nutricio en ácido fólico, vitaminas B6 y B12.

Homocysteine is a thiol-containing amino acid derived from methionine metabolism that can be degraded through two enzymatic pathways: remethylation and trans-sulfuration. In remethylation, homocysteine regenerates methionine. In the trans-sulfuration pathway, homocysteine forms cysteine. Due to the rapid metabolic utilization, the plasma concentration of this amino acid is low. Homocysteine circulates as free thiol, homocystine, or bound to free cysteine or to cysteine residues of proteins. Genetic defects of some enzymes in the homocysteine metabolism, or nutritional deficiencies of folic acid, vitamin B6 and B12 lead to an increase in homocysteine plasma concentration and is associated to an increment in cardiovascular diseases. On the basis of clinical and epidemiological studies, homocysteine plasma concentration is considered to be an independent risk factor for the development of atherothrombotic and cardiovascular diseases. The present review describes the homocysteine metabolism, the epidemiological evidence showing the association between homocysteine and the incidence of cardiovascular diseases. The mechanisms by which homocysteine produces vascular damage are indicated. Finally, some recommendations are given for the nutritional therapy of patients with hyperhomocysteinemia.

Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland.

During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.

Amino acid intake during lactation and amino acids of plasma and human milk.

The aim of this study was to determine the free amino acid pool in plasma and milk in marginally nourished lactating women. Twenty-eight rural women (age, 23.9+/-5y; weight 50.2+/-4.9 kg; height, 148.2+/-4.8 cm) were studied under metabolic balance conditions. Subjects were divided into 6 groups (5-6 women in each), representing rural mothers postweaning and in the 15, 3rd, and 6th months of lactation; nonpregnant, nonlactating controls were from rural and urban areas. Amino acid analyses of diet and of plasma and milk samples were performed using a Beckman 6300 amino acid analyzer. Lysine intakes were lower than the recommended intake for lactating women (RDA). Plasma amino acid profiles differed between the lactating and weaned groups: aspartate and isoleucine increased at the 6th month (P < 0.05), while valine declined over weaning time (P < 0.05). In milk, valine and proline decreased at the 6th month (P < 0.05), while serine rose at the 3rd month. Free amino acid pools were 1- to 15-fold higher in plasma than in milk for branched-chain amino acids and basic, aromatic, and neutral amino acids. In mammary tissue these amino acids can be channeled to tissue and milk protein synthesis or to catabolic pathways. Glutamate was 40-fold higher in milk with respect to plasma content. This was the predominant amino acid in the free amino acid pool in milk. These results suggest selective amino acid transport in mammary tissue during lactation.

Induction of expression of branched-chain aminotransferase and alpha-keto acid dehydrogenase in rat tissues during lactation.

This study was designed to determine the effect of lactation and weaning on the gene expression of branched-chain aminotransaminase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD) in different tissues of the lactating rat. BCAT activity increased in mammary tissue during lactation and was 6-fold higher than in virgin rats. This increase was associated with an increase in protein levels measured by immunoblot analysis, and with an increase in BCAT mitochondrial (BCATm) mRNA concentration. Twenty-four hours after weaning, BCAT activity, protein concentration, and mRNA levels in the dam decreased. BCAT activity, protein enzyme levels, and BCATm mRNA concentration in muscle were higher in weaning rats than in lactating rats. BCAT cytosolic (BCATc) mRNA was not expressed in mammary tissue, and there was no BCATc enzyme detected by Western blot in any physiological state. Mammary tissue BCKD activity increased and was active (dephosphorylated) during the lactation period. The level of enzyme also increased and the mRNA level for the E2 subunit in mammary tissue was 10-fold higher than the virgin values. Hepatic enzyme activity increased during weaning, and this was associated with the protein level and with the mRNA level of the E2 subunit. Muscle BCKD activity and protein content were the lowest of all tissues, and the E2 subunit mRNA level was barely detected by Northern blot analysis. The results suggest gene regulation of the two main catabolic enzymes of the branched-chain amino acid metabolism during lactation.

Use of sulfhydryl reagents to investigate branched chain alpha-keto acid transport in mitochondria.

The goal of this paper was to determine the contribution of the mitochondrial branched chain aminotransferase (BCATm) to branched chain alpha-keto acid transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data suggest that essential cysteine residues in BCATm are accessible from the cytosolic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimide (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in reconstituted mitochondrial detergent extracts by 70% and in the intact organelle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzyme. Additionally, the apparent mitochondrial equilibrium KIC concentration was decreased 50-60% after NEM labeling, and this difference could not be attributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix aspartate and glutamate levels revealed that the effects of NEM on the steady-state KIC concentration resulted from inhibition of BCATm catalyzed transamination of KIC with matrix glutamate to form leucine. Furthermore, circular dichroism spectra of recombinant human BCATm with liposomes showed that the commercial lipids used in the reconstituted transport assay contain BCAT amino acid substrates. Thus BCATm is distinct from the branched chain alpha-keto acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact and reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates.

Characterization of methylaminoisobutyric acid transport by system A in rat mammary gland.

During lactation, the mammary gland has a large demand for amino acids for the synthesis of milk proteins and fatty acids. Arteriovenous differences in amino acids across the mammary gland show an elevated uptake of small neutral amino acids that are mainly transported via system A. The purpose of this study was to characterize the transport of methylaminoisobutyric acid (MeAIB), an amino acid analog used to model transport by system A in lactating rat mammary gland explants. MeAIB accumulation in mammary gland cells increased steadily, and after 3 hours of incubation, the intracellular concentration of the analog was 8-fold higher than the concentration in the medium. MeAIB transport into mammary gland explants showed a Km of 3.3 +/- 0.4 mmol/L and a maximal velocity (Vmax) of 555 +/- 23 pmol/microL intracellular fluid (ICF) x min, indicating a system with high capacity but low affinity for its substrate. MeAIB transport into mammary tissue depended highly on Na+, and the uptake was inhibited by addition of natural and analog small neutral amino acids. Cationic, anionic, and large neutral amino acids did not reduce MeAIB transport into mammary gland explants. Preincubation of mammary gland explants in an amino acid-free medium stimulated MeAIB transport, suggesting an adaptive regulation. The addition of an equimolar mixture of alanine, glycine, and serine to the preincubation medium inhibited stimulation of MeAIB transport. Furthermore, stimulation of MeAIB uptake by amino acid starvation was also prevented by the addition of actinomycin D, cycloheximide, tunicamycin, and colchicine. Dibutyryl cyclic adenosine monophosphate (cAMP) increased MeAIB uptake, whereas phorbol 12-myristate 13-acetate (PMA) did not stimulate MeAIB transport. During the first postweaning days, kinetic analyses showed a decrease of 27% in the Vmax. Injection of rat lactating mammary gland mRNA into Xenopus laevis oocytes induced expression of the MeAIB transport system; however, the induction was only 83% above background MeAIB uptake. The results of this study provide a partial explanation for the formation of the metabolic pool of small neutral amino acids in the lactating mammary gland.

Preeclampsia is associated with low circulating levels of insulin-like growth factor I and 1,25-dihydroxyvitamin D in maternal and umbilical cord compartments.

Insulin-like growth factor I (IGF-I) stimulates renal and placental 1,25-dihydroxyvitamin D [1,25-(OH)2D] and is considered an important regulator of fetal growth. As 1,25-(OH)2D and birth weight are low in preeclampsia, this study was undertaken to determine whether circulating levels of IGF-I were associated with serum 1,25-(OH)2D concentrations in preeclamptic (PE group) and normotensive (NT group) pregnancies. Maternal and umbilical cord serum levels of IGF-I and 1,25-(OH)2D were significantly (P < 0.01) lower in the PE group than in the NT group. The concentrations of these two hormones correlated significantly in the umbilical cord (P < 0.05) and in the maternal (P < 0.001) compartments of the PE and NT groups, respectively. The amount of IGFBP-3 was 64% lower whereas that of IGFBP-1 was 2.9-fold higher in umbilical cord serum of the PE group compared with the NT group. In addition, maternal and umbilical cord serum IGF-I correlated significantly (P < 0.05) with weight and length at birth only in the PE group. In conclusion, the results of this study indicate that circulating IGF-I and 1,25-(OH)2D levels in both maternal and umbilical cord compartments are low in preeclampsia. Furthermore, this study suggests a differential regulatory effect of IGF-I on 1,25-(OH)2D synthesis and fetal growth depending on the presence or absence of preeclampsia.

Hepatic histidase and muscle branched chain aminotransferase gene expression in experimental nephrosis.

Protein and amino acid metabolism is altered during nephrotic syndrome. However, the expression of the amino acid degrading enzymes has not been well studied. The objective of this work was to assess the expression of hepatic histidase (Hal) and skeletal muscle mitochondrial branched chain amino transferase (BCATm) in rats with experimental nephrotic syndrome induced by a single injection of puromycin aminonucleoside (150 mg/kg). Six days after the injection rats were killed and hepatic Hal and skeletal muscle BCATm activities were measured. Also, total mRNA from both tissues was isolated and Hal and BCATm mRNA expression were analyzed by Northern blot. Rats with NS showed a reduction in food intake with respect to the control group. Hepatic Hal activity increased significantly in nephrotic and pair fed rats by 59% compared to control group. This change in activity was associated with a corresponding increase in Hal mRNA abundance. On the other hand, skeletal muscle BCATm activity and mRNA abundance were similar in the three groups studied. These results suggest that the increase in Hal expression was associated with the reduced food intake and not to the NS. However, BCAT expression did not change indicating the importance of BCAA in body nitrogen conservation.

Histidine-imbalanced diets stimulate hepatic histidase gene expression in rats.

A high protein concentration in the diet induces the gene expression of several amino acid degrading enzymes such as histidase (Hal) in rats. It is important to understand whether the amino acid pattern of the dietary protein affects the gene expression of these enzymes. The purpose of the present work was to study the effect of a histidine-imbalanced diet on the activity and mRNA concentration of rat hepatic histidase. Seven groups of six rats were fed one of the following diets: 1) 6% casein (basal), 2) 20% casein, 3) 35% casein, 4) an imbalance diet containing 6% casein plus a mixture of indispensable amino acids (IAA) equivalent to a 20% casein diet without histidine (I-20), 5) 6% casein plus a mixture of IAA equivalent to a 35% casein diet without histidine (I-35), 6) a corrected diet containing 6% casein plus IAA including histidine equivalent to a 20% casein diet, 7) a corrected diet containing 6% casein plus IAA including histidine equivalent to a 35% casein diet. Serum histidine concentration was inversely proportional to the protein content of the diet, and it was significantly higher in rats fed the corrected diets compared to their respective imbalanced diet groups. Hal activity increased as the protein content of the diet increased. Greater histidine imbalance resulted in lower food intake and higher Hal activity. Rats fed histidine-corrected diets had lower activity than their respective imbalanced groups. Differences in Hal activity were associated with differences in the concentration of Hal mRNA. These results indicate that rats fed a histidine-imbalanced diet exhibit reduced food intake and weight gain and increased Hal gene expression as a consequence of an increased amino acid catabolism.