Sugar sensing by enterocytes combines polarity, membrane bound detectors and sugar metabolism

Sugar consumption and subsequent sugar metabolism are known to regulate the expression of genes involved in intestinal sugar absorption and delivery. Here we investigate the hypothesis that sugar-sensing detectors in membranes facing the intestinal lumen or the bloodstream can also modulate intestinal sugar absorption. We used wild-type and GLUT2-null mice, to show that dietary sugars stimulate the expression of sucrase-isomaltase (SI) and L-pyruvate kinase (L-PK) by GLUT2-dependent mechanisms, whereas the expression of GLUT5 and SGLT1, did not rely on the presence of GLUT2. By providing sugar metabolites, sugar transporters, including GLUT2, fuelled a sensing pathway. In Caco2/TC7 enterocytes, we could disconnect the sensing triggered by detector from that produced by metabolism, and found that GLUT2 generated a metabolism-independent pathway to stimulate the expression of SI and L-PK. In cultured enterocytes, both apical and basolateral fructose could increase the expression of GLUT5, conversely, basolateral sugar administration could stimulate the expression of GLUT2. Finally, we located the sweet-taste receptors T1R3 and T1R2 in plasma membranes, and we measured their cognate G alpha Gustducin mRNA levels. Furthermore, we showed that a T1R3 inhibitor altered the fructose-induced expression of SGLT1, GLUT5, and L-PK. Intestinal gene expression is thus controlled by a combination of at least three sugar-signaling pathways triggered by sugar metabolites and membrane sugar receptors that, according to membrane location, determine sugar-sensing polarity. This provides a rationale for how intestine adapts sugar delivery to blood and dietary sugar provision.     

Secondary phloem anatomy of Cycadeoidea (Bennettitales)

Secondary phloem anatomy of several species of Cycadeoidea is described from trunks in the Wieland Collection, Peabody Museum of Natural History. The trunks were collected from the Lakota Formation, Lower Cretaceous, Black Hills of South Dakota. Secondary phloem is extensively developed and consists of alternating, tangential bands of fibers and sieve elements, with rare phloem parenchyma. Uniseriate rays, 2-22 cells high, occur between every one to three files of the axial system. Fibers are long, more than 1200 μm, approximately 26.6-34.2 μm in diameter, and have slit-like apertures on the lateral walls. Sieve elements range from 16-25 μm in diameter and are up to 500 μm long. Elliptical sieve areas appear on both end and radial walls and measure 10 μm across; minute spots, which may represent sieve pores, are present within the sieve areas. Secondary phloem of North American Cycadeoidea is similar in organization (alternating tangential bands) and cell types (sieve cells, fibers, axial parenchyma) to that known in other extant and fossil cycadophytes and some seed ferns. The unusual pattern of cell types and thickness of secondary phloem is discussed in the context of plant habit, phloem efficiency, and potential phylogenetic importance.     

Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

The mouse arylamine N-acetyltransferase 2 (Nat2) and its homologue (NAT1) in humans are known to detoxify xenobiotic arylamines and are also thought to play a role in endogenous metabolism. Human NAT1 is highly over-expressed in estrogen receptor positive breast tumours and is implicated in susceptibility to neural tube defects. In vitro assays have suggested an endogenous role for human NAT1 in folate metabolism, but in vivo evidence to support this hypothesis has been lacking. Mouse Nat2 provides a good model to study human NAT1 as it shows similar expression profiles and substrate specificities. We have generated transgenic mice lacking a functional Nat2 gene and compared the urinary levels of acetylated folate metabolite para-aminobenzoylglutamate in Nat2 knockout and Nat2 wild-type mice. These results support an in vivo role for mouse Nat2/human NAT1 in folate metabolism. In addition, effects of the Nat2 deletion on sex ratios and neural tube development are described.     

PduL is an evolutionarily distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B(12)-dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His(8)) was purified, and its kinetic constants were determined: the V(max) was 51.7 +/- 7.6 micromol min(-1) mg(-1), and the K(m) values for propionyl-PO(4)(2-) and acetyl-PO(4)(2-) were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.     

NM23-H2, an estrogen receptor beta-associated protein, shows diminished expression with progression of atherosclerosis

While estrogen receptor (ER) profile plays an important role in response to estrogens, receptor coregulators act as critical determinants of signaling. Although the clinical effects of ovarian hormones on various normal and pathological processes are an active area of research, the exact signaling effects on, for example, the vessel wall, are incompletely understood. Hence, we sought to discover proteins that associate with ERbeta, the isoform that shows upregulated mRNA expression after arterial injury. Using a yeast two-hybrid screen we identified NM23-H2, a multifaceted metastasis suppressor candidate protein, as an ERbeta-associated protein. Although NM23-H2 was immunodetected in arteries from young subjects (27 +/- 6 yr, 14 men and 6 women) with benign intimal hyperplasia, expression was diminished in fatty streaks/atheromas and altogether absent in advanced atherosclerotic lesions. Both nm23-H2 mRNA and protein were expressed by vascular cells in vitro. Treatment with 17beta-estradiol and an ERbeta-selective agonist, diarylpropionitrile, increased protein expression of NM23-H2; an effect that was not seen with an ERalpha-selective agonist, propylpyrazole-triol. Estrogen also prompted nuclear localization of NM23-H2 protein in human coronary smooth muscle cells (SMCs). An in vitro mimic of inflammation decreased the expression of NM23-H2 in SMCs, which was restored on addition of estrogen and dependent on the estrogen receptor. In summary, we report the novel association of NM23-H2 with ERbeta and show for the first time its expression in vascular cells and demonstrate regulation of its expression and localization by estrogen. In that the abundance of NM23-H2 diminishes with both the advancement of atherosclerosis and inflammation, this ERbeta-associated protein may play an important role in mediating the vasculoprotective effects of estrogens.     

Inhibition of mycobacterial arylamine N-acetyltransferase contributes to anti-mycobacterial activity of Warburgia salutaris

In this study, we show that extracts and a purified compound of Warburgia salutaris exhibit anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG Pasteur. The extracts did not inhibit growth of Escherichia coli and were not toxic to cultured mammalian macrophage cells at the concentrations at which anti-mycobacterial activity was observed. The extract and pure compound inhibited pure recombinant arylamine N-acetyltransferase (NAT), an enzyme involved in mycobacterial cell wall lipid synthesis. Moreover, neither extract nor pure compound inhibited growth of a strain of M. bovis BCG in which nat has been deleted suggesting that NAT may indeed be a target within the mycobacterial cell. The purified compound is a novel drimane sesquiterpenoid lactone, 11alpha-hydroxycinnamosmolide. These studies show that W. salutaris is a useful source of anti-tubercular compounds for further analysis and supports the hypothesis of a link between NAT inhibition and anti-mycobacterial activity.     

Zinc-(II) 2,9,16,23-tetrakis (methoxy) phthalocyanine: potential photosensitizer for use in photodynamic therapy in vitro

Photodynamic therapy (PDT) is an innovative treatment for several types of malignant and non-malignant disease. In the present study, ZnPcOCH(3) was investigated on a human larynx-carcinoma cell line (Hep-2) for its use in PDT. This drug exhibited favourable properties as a photosensitizer in vitro because ZnPcOCH(3) is able to penetrate efficiently in the cytoplasm of cultured cancer cells and is partially localized in lysosomes. The results show that ZnPcOCH(3)-PDT-induced apoptosis by caspase dependent pathway. The new compound shows a good photosensitizing efficiency in vitro on Hep-2 cells, encouraging further in vivo studies.     

Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.     

Effectiveness of imazalil to control the effect of fungal deterioration on mummies at the Mexico City Museum "El Carmen"

We present a study on the control and elimination of the fungi affecting the mummies specifically at the museum "El Carmen", in San Angel, Mexico City. Twelve analysed mummies presented an important deterioration attributed to colonizing fungi. The degree of fungal contamination and the efficacy of imazalil were evaluated. Two samplings were performed in order to isolate and identify the fungal genera, one for control and the other after the treatment. Isolation was done by the carpet-square technique and identification was performed by morphological features. Each sampling gave a total of 100 samples as follows: 17 from the air, 23 from the walls and 60 from the mummies. Samples were cultured on Sabouraud dextrose agar. From the first sampling a total of 649 colonies corresponding to 24 genera were obtained being the most frequent Penicillium, Cladophialophora and Aspergillus. From the second sampling, after the imazalil treatment, which was applied by means of lit candles containing the antifungal drug, 57 colonies were recovered, representing a 91.2% fungal reduction; 18 genera were eliminated. In spite of resistance showed by many Penicillium strains, the imazalil is an alternative drug for the control of fungal colonization on these studied materials.     

Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists

The identification of the antigen recognition receptors for innate immunity, most notably the Toll-like receptors, has sparked great interest in therapeutic manipulation of the innate immune system. Toll-like receptor agonists are being developed for the treatment of cancer, allergies and viral infections, and as adjuvants for potent new vaccines to prevent or treat cancer and infectious diseases. As recognition grows of the role of inappropriate Toll-like receptor stimulation in inflammation and autoimmunity, significant efforts have begun to develop antagonists to Toll-like receptors as well.