Kinetic analysis of L-glutamine transport into porcine jejunal enterocyte brush-border membrane vesicles

L-Glutamine transport into porcine jejunal enterocyte brush border membrane vesicles was studied. Uptake was mediated by a Na(+)-dependent and a Na(+)-independent pathway as well as by diffusion. The initial rates of glutamine uptake over a range of concentrations is both Na(+)-gradient and Na(+)-free conditions were analyzed and kinetic parameters were obtained. Na(+)-dependent glutamine transport had a K(m) of 0.77 +/- 0.16 mM and a Jmax of 70.7 +/- 5.8 pmol mg protein-1 s-1; Na(+)-independent glutamine transport had a K(m) of 3.55 +/- 0.78 mM and a Jmax of 55.1 +/- 6.6 pmol mg protein-1 s-1. The non-saturable component measured with HgCl2-poisoned brush border membrane vesicles in the Na(+)-free condition contained passive diffusion and non-specific membrane binding and was defined to be apparent glutamine diffusion and the glutamine permeability coefficient (Kdiff) was estimated to be Kdiff = 3.78 +/- 0.06 pmol 1 mg protein-1 mmol-1 s-1. Results of inhibition experiments showed that Na(+)-dependent glutamine uptake occurred primarily through the brush border system-B degree transporters, whereas Na(+)-independent glutamine uptake occurred via the system-L transporters. Furthermore, the kinetics of L-leucine and L-cysteine inhibition of L-glutamine uptake demonstrated that neutral amino acids sharing the same brush border transporters can effectively inhibit each other in their transport.     

Complete mitochondrial genome of the Thai Red Junglefowl(Gallus gallus) and phylogenetic analysis

正DEAR EDITOR,In this study,we sequenced the complete mitochondrial genome(mitogenome)of the Thai Red Junglefowl(RJF;Gallus gallus)using the next-generation sequencing(NGS)platform of the Ion Torrent PGM.Samples were taken from Mae Wang District,Chiang Mai Province,northern Thailand.Our data showed the complete mitogenome to be 16 785bp in length,composed by 13 protein-coding genes,22tRNA genes,two rRNA genes,and one control region.The genome nucleotide composition was 30.3%A,23.7%T,32.5%C,and 13.5%G,resulting in a high percentage of A+T     

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 μm diameter and 150 μm depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 μm in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.Open in a separate windowClick here to view.^{(45M, flv)}     

Dietary Fat Content and Fiber Type Modulate Hind Gut Microbial Community and Metabolic Markers in the Pig

Obesity leads to changes in the gut microbial community which contribute to the metabolic dysregulation in obesity. Dietary fat and fiber affect the caloric density of foods. The impact of dietary fat content and fiber type on the microbial community in the hind gut is unknown. Effect of dietary fat level and fiber type on hindgut microbiota and volatile fatty acid (VFA) profiles was investigated. Expression of metabolic marker genes in the gut, adipose tissue and liver was determined. A 2×2 experiment was conducted in pigs fed at two dietary fat levels (5% or 17.5% swine grease) and two fiber types (4% inulin, fermentable fructo-oligosaccharide or 4% solka floc, non-fermentable cellulose). High fat diets (HFD) resulted in a higher (P<0.05) total body weight gain, feed efficiency and back fat accumulation than the low fat diet. Feeding of inulin, but not solka floc, attenuated (P<0.05) the HFD-induced higher body weight gain and fat mass accumulation. Inulin feeding tended to lead to higher total VFA production in the cecum and resulted in a higher (P<0.05) expression of acyl coA oxidase (ACO), a marker of peroxisomal β-oxidation. Inulin feeding also resulted in lower expression of sterol regulatory element binding protein 1c (SREBP-1c), a marker of lipid anabolism. Bacteria community structure characterized by DGGE analysis of PCR amplified 16S rRNA gene fragments showed that inulin feeding resulted in greater bacterial population richness than solka floc feeding. Cluster analysis of pairwise Dice similarity comparisons of the DGGE profiles showed grouping by fiber type but not the level of dietary fat. Canonical correspondence analysis (CCA) of PCR- DGGE profiles showed that inulin feeding negatively correlated with back fat thickness. This study suggests a strong interplay between dietary fat level and fiber type in determining susceptibility to obesity.     

A role for autophagic protein beclin 1 early in lymphocyte development

Autophagy is a highly regulated and evolutionarily conserved process of cellular self-digestion. Recent evidence suggests that this process plays an important role in regulating T cell homeostasis. In this study, we used Rag1(-/-) (recombination activating gene 1(-/-)) blastocyst complementation and in vitro embryonic stem cell differentiation to address the role of Beclin 1, one of the key autophagic proteins, in lymphocyte development. Beclin 1-deficient Rag1(-/-) chimeras displayed a dramatic reduction in thymic cellularity compared with control mice. Using embryonic stem cell differentiation in vitro, we found that the inability to maintain normal thymic cellularity is likely caused by impaired maintenance of thymocyte progenitors. Interestingly, despite drastically reduced thymocyte numbers, the peripheral T cell compartment of Beclin 1-deficient Rag1(-/-) chimeras is largely normal. Peripheral T cells displayed normal in vitro proliferation despite significantly reduced numbers of autophagosomes. In addition, these chimeras had greatly reduced numbers of early B cells in the bone marrow compared with controls. However, the peripheral B cell compartment was not dramatically impacted by Beclin 1 deficiency. Collectively, our results suggest that Beclin 1 is required for maintenance of undifferentiated/early lymphocyte progenitor populations. In contrast, Beclin 1 is largely dispensable for the initial generation and function of the peripheral T and B cell compartments. This indicates that normal lymphocyte development involves Beclin 1-dependent, early-stage and distinct, Beclin 1-independent, late-stage processes.     

mRNA Expression Profiles for Prostate Cancer following Fractionated Irradiation Are Influenced by p53 Status

We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy × 10, 1 Gy × 10, and 2 Gy × 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 × 10^-73, 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 × 10^-30) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.     

Genetic Architecture Underlying Nascent Speciation—The Evolution of Eurasian Pigs under Domestication

Speciation is a process whereby the evolution of reproductive barriers leads to isolated species. Although many studies have addressed large-effect genetic footprints in the advanced stages of speciation, the genetics of reproductive isolation in nascent stage of speciation remains unclear. Here, we show that pig domestication offers an interesting model for studying the early stages of speciation in great details. Pig breeds have not evolved the large X-effect of hybrid incompatibility commonly observed between “good species.” Instead, deleterious epistatic interactions among multiple autosomal loci are common. These weak Dobzhansky–Muller incompatibilities confer partial hybrid inviability with sex biases in crosses between European and East Asian domestic pigs. The genomic incompatibility is enriched in pathways for angiogenesis, androgen receptor signaling and immunity, with an observation of many highly differentiated cis-regulatory variants. Our study suggests that partial hybrid inviability caused by pervasive but weak interactions among autosomal loci may be a hallmark of nascent speciation in mammals.     

Low-dose photons modify liver response to simulated solar particle event protons

The health consequences of exposure to low-dose radiation combined with a solar particle event during space travel remain unresolved. The goal of this study was to determine whether protracted radiation exposure alters gene expression and oxidative burst capacity in the liver, an organ vital in many biological processes. C57BL/6 mice were whole-body irradiated with 2 Gy simulated solar particle event (SPE) protons over 36 h, both with and without pre-exposure to low-dose/low-dose-rate photons ((57)Co, 0.049 Gy total at 0.024 cGy/h). Livers were excised immediately after irradiation (day 0) or on day 21 thereafter for analysis of 84 oxidative stress-related genes using RT-PCR; genes up or down-regulated by more than twofold were noted. On day 0, genes with increased expression were: photons, none; simulated SPE, Id1; photons + simulated SPE, Bax, Id1, Snrp70. Down-regulated genes at this same time were: photons, Igfbp1; simulated SPE, Arnt2, Igfbp1, Il6, Lct, Mybl2, Ptx3. By day 21, a much greater effect was noted than on day 0. Exposure to photons + simulated SPE up-regulated completely different genes than those up-regulated after either photons or the simulated SPE alone (photons, Cstb; simulated SPE, Dctn2, Khsrp, Man2b1, Snrp70; photons + simulated SPE, Casp1, Col1a1, Hspcb, Il6st, Rpl28, Spnb2). There were many down-regulated genes in all irradiated groups on day 21 (photons, 13; simulated SPE, 16; photons + simulated SPE, 16), with very little overlap among groups. Oxygen radical production by liver phagocytes was significantly enhanced by photons on day 21. The results demonstrate that whole-body irradiation with low-dose-rate photons, as well as time after exposure, had a great impact on liver response to a simulated solar particle event.     

Isolation and photodynamic effects of hematoporphyrin derivative components: a chromatographic analysis of the starting materials

Twenty different fractions of hematoporphyrin derivatives (HpD) and eight fractions of an HpD dimer mixture were isolated utilizing isocratic reversed-phase ion-pair high-performance liquid chromatography. These fractions were characterized by UV-visible and fluorescence spectrophotometry. Fluorescence quantum yields and photokill efficiency for each fraction in PTK₂ epithelial cells were obtained. Results indicate that some part of the photoactivity exhibited by HpD may be due to impurities present in the HpD starting material, hematoporphyrin-IX dihydrochloride, depending on its source. It was also found that hematoporphyrin D, a commercial acetylated product formed during synthesis of HpD, contained a higher percentage of monomers than would be expected.