A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Fibronectin-independent attachment of human gingival fibroblasts to interstitial and basement membrane collagens

We have studied the ability of human gingival fibroblasts (HGF) to attach to different interstitial (types I, II and III) and basement membrane (types IV and V) collagens. HGF cells were plated onto collagen-coated Petri dishes under various conditions and the percentage of cells attaching to the collagen was determined. HGF were found to attach to all the different types of native collagens, but attached poorly to the corresponding denatured collagens. When plated in the presence of 15% fetal bovine serum (FBS) or fibronectin-depleted FBS, similar percentages (approximately 85%) of cells attached to both interstitial and basement membrane collagens, demonstrating an attachment mechanism that is independent of plasma fibronectin. That the attachment in the presence of serum was also independent of cellular fibronectin was shown by the inability of fibronectin antibodies to block attachment to any of the collagen types. HGF were also capable of attaching to all of the collagen types in the complete absence of serum. In previous studies, investigators using cell lines have suggested that cell attachment in the absence of serum is non-physiological. However, the serum-free attachment of HGF to collagen was found to be dependent on cellular protein synthesis indicating that this attachment mechanism has biological significance.     

Bifidobacterium longum supplementation improves age-related delays in fracture repair

Age-related delays in bone repair remains an important clinical issue that can prolong pain and suffering. It is now well established that inflammation increases with aging and that this exacerbated inflammatory response can influence skeletal regeneration. Recently, simple dietary supplementation with beneficial probiotic bacteria has been shown to influence fracture repair in young mice. However, the contribution of the gut microbiota to age-related impairments in fracture healing remains unknown. Here, we sought to determine whether supplementation with a single beneficial probiotic species, Bifidobacterium longum (B. longum), would promote fracture repair in aged (18-month-old) female mice. We found that B. longum supplementation accelerated bony callus formation which improved mechanical properties of the fractured limb. We attribute these pro-regenerative effects of B. longum to preservation of intestinal barrier, dampened systemic inflammation, and maintenance of the microbiota community structure. Moreover, B. longum attenuated many of the fracture-induced systemic pathologies. Our study provides evidence that targeting the gut microbiota using simple dietary approaches can improve fracture healing outcomes and minimize systemic pathologies in the context of aging.     

The distinct nucleotide binding states of the transporter associated with antigen processing (TAP) are regulated by the nonhomologous C-terminal tails of TAP1 and TAP2.

The transporter associated with antigen processing (TAP) delivers peptides into the lumen of the endoplasmic reticulum for binding onto major histocompatibility complex class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each with an N-terminal transmembrane domain and a C-terminal cytosolic nucleotide binding domain (NBD). The two NBDs have distinct intrinsic nucleotide binding properties. In the resting state of TAP, the NBD1 has a much higher binding activity for ATP than the NBD2, while the binding of ADP to the two NBDs is equivalent. To attribute the different nucleotide binding behaviour of NBD1 and NBD2 to specific sequences, we generated chimeric TAP1 and TAP2 polypeptides in which either the nonhomologous C-terminal tails downstream of the Walker B motif, or the core NBDs which are enclosed by the conserved Walker A and B motifs, were reciprocally exchanged. Our biochemical and functional studies on the different TAP chimeras show that the distinct nucleotide binding behaviour of TAP1 and TAP2 is controlled by the nonhomologous C-terminal tails of the two TAP chains. In addition, our data suggest that the C-terminal tail of TAP2 is required for a functional transporter by regulating ATP binding. Further experiments indicate that ATP binding to NBD2 is important because it prevents simultaneous uptake of ATP by TAP1. We propose that the C-terminal tails of TAP1 and TAP2 play a crucial regulatory role in the coordination of nucleotide binding and ATP hydrolysis by TAP.     

Genetic assessment of the internal transcribed spacer region (ITS1.2) in Mangifera indica L. landraces

Mango (Mangifera indica) is one of the most important tropical fruits in the world. Twenty-two genotypes of native mangoes from different regions of southern Iran (Hormozgan and Kerman) were collected and analyzed for the ribosomal genes. GC content was found to be 55.5%. Fu and Li’s D* test statistic (0.437), Fu and Li’s F* test statistic (0.500) and Tajima’s D (1.801) were positive and nonsignificant. A total of 769 positions were identified (319 with insertion or deletion including 250 polymorphic and 69 monomorphic loci; 450 loci without any insertion or deletion including 35 Singletons and 22 haplotypes). Nucleotide diversity of 0.309 and a high genetic differentiation including Chi square of 79.8; P value of 0.3605 and df value of 76 was observed among mango genotypes studied. The numerical value of the ratio dN/dS (0.45) indicated a pure selection in the examined gene and the absence of any key changes. Cluster analysis differentiated the mango used in this research (M. indica L.) into two genotypes but could not differentiate their geographical locations. The results of this study indicated that a high genetic distance exists between HajiGholam (Manojan) and Arbabi (Rodan) genotypes and showed higher genetic diversity in mango of Rodan region. Results of present study suggested that for successful breeding, the genotypes of Rodan region mango especially Arbabi mango can be used as a gene donor and ITS can be a suitable tool for genetic evaluations of inter and intra species.     

Efficient,Cyanine Dye Based Bilayer Solar Cells

Simple bilayer solar cells, using commercially available cationic cyanine dyes as donors and evaporated C₆₀ layer as an acceptor are prepared. Cyanine dyes with absorption maxima of 578, 615 and 697 nm having either perchlorate or hexafluorophosphate counter‐ions are evaluated. The perchlorate dye leads to cells with S‐shape current‐voltage curves; only the dyes with the hexafluorophosphate counter‐ions lead to efficient solar cells. When the wide bandgap dyes are employed, S‐shape current‐voltage curves are obtained when the conductive polymer PEDOT:PSS is used as hole transport layer. Substitution of PEDOT:PSS with MoO₃ leads to cells with more rectangular current–voltage curves and high fill factors. Additionally, the cells using the MoO₃ layer for hole extraction lead to high open circuit voltages of 0.9 V. In the case that a low bandgap hexafluorophosphate dye is used with the HOMO above that of the PEDOT:PSS the cell performance is independent on the type of hole transport layer employed. Using this approach, bilayer solar cells are obtained with power efficiencies ranging from 1.8 to 2.9% depending on the particular dye employed. These are impressive numbers for bilayer solar cell that are partially solution processed in ambient conditions.     

Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine

The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca²⁺ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca²⁺ uptake completion prevented this response. Maurocalcine enhanced equilibrium [³H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent K_i for Mg²⁺ inhibition of [³H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca²⁺] from 10 μM to 0.1 μM or at cytoplasmic [Mg²⁺] ≥ 30 μM. At 0.1 μM [Ca²⁺], only channels that displayed poor activation by Ca²⁺ were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.     

Phenotypic and Genotypic Identification of the Most Acidifiers LAB Strains Isolated from Fermented Food

Biology Bulletin - Over 18 strains of lactic acid bacteria “LAB” isolated from different traditionally fermented products mainly sourdough and fermented vegetables, have been...