Metabolic variants of intestinal alkaline phosphatase in relation to fat absorption: in situ demonstration with the organ-specific inhibitors l-phenylalanine and l-homoarginine

Summary The localization of rat intestinal alkaline phosphatase has been studied in relation to fat absorption. The observations support a theory of conversion, within the intestinal mucosa, of intestinal type to liver type alkaline phosphatase when the criterion of differential sensitivity to two amino acid inhibitors, l-phenylalanine, and l-homoarginine, is applied.Following a three hour in vivo exposure to mixtures of oleic acid, sodium taurocholate, and lauric acid, the epithelium becomes depleted of its l-phenylalanine-sensitive, intestinal type alkaline phosphatase. At the same time, enriched activity is seen in the lamina propria; this activity is both particulate and diffuse, and is present both in the connective tissue matrix and in cells, including macrophages, eosinophils and lymphocytes. Most of this enzyme is inhibited by l-homoarginine, a property characteristic of liver type alkaline phosphatase.The localization of enzyme-positive particles 0.5 to 1.0 in diameter in both epithelium and lamina propria appears identical to that of particulate fat. A physical association between transport of absorbed fat and metabolic conversion of intestinal type alkaline phosphatase is postulated.This work was aided in part by grants-in-aid [CA-3332-01, K6-CA-18,453] from the National Cancer Institute, National Institutes of Health, U.S.P.H.S. and the John A. Hartford Foundation, Inc.Pre-doctoral trainee, U.S.P.H.S. grant GM01451.Holder of a Juan Marsh Foundation Fellowship, Lemuel Shattuck Hospital.     

Thymus cell maturation: II. Differentiation of three “mature” subclasses in Vivo

Several thymus cell subclasses may be defined on the basis of their sedimentation velocity, their light-scattering properties (a measure of cell volume), or binding of a fluoresceinated anti-Thy 1.2 antiserum. Using a multiparameter fluorescence-activated cell sorter (FACS), cells with distinguishable light-scattering or fluorescence intensity (after staining with fluorescein anti-Thy 1.2) were separable for analysis of intrathymic maturation pathways. Outer thymic cortical large and medium lymphocytes were the only cells labeled within 1 hr after transcapsular diffusion of administered [³H]thymidine. These labeled cells were also entirely contained in the brightest fluorescence intensity (with fluorescein anti-Thy 1.2) subclass. Under conditions of [¹H] thymidine “chase” in vivo, label shifted proportionately and apparently in parallel to three “mature” subclasses: (1) small thymocytes with high surface concentrations of Thy 1.2, representing ~ 80% of all thymus cells; (2) slightly larger cells, with very low surface Thy 1.2, which are indistinguishable from cortisone-resistant thymocytes, and which make up less than 10% of all thymus cells; (3) dead or fragile cells.     

Possible sites of release of neurosecretory granules in the sinus gland of the crayfish,Orconectes nais

Summary The sinus gland is a neurohemal organ located in the crayfish eyestalk and represents a storage site for neurohormones prior to their release into the circulation. The sinus gland contains three classes of electron dense, membrane-limited granules. Class 3 granules are the largest and most electron dense of the granules found in the sinus gland. Granules of class 1 are the smallest, while those of class 2 are the most abundant. Generally, all granules undergo similar changes during their release.Release of neurosecretory material may be initiated by a preliminary fragmentation of the parent granules into smaller granules. Following the formation of numerous smaller granules, these move to the plasma membrane and their limiting membrane apparently fuses with it thus releasing its contents into the external lamina which is applied to the sinusoidal surface of the axon terminals. Granule release does not appear to occur along the entire plasma membrane adjacent to the blood sinus but, instead, probably occurs only at specific active sites on the membrane. The active sites are characterized in part by an accumulation of small granules and clear vesicles against the cytoplasmic side of the plasma membrane. At the site of release of the neurohormone, there is often an accumulation of dense homogeneous material beneath the axolemma.Occasionally, axon endings filled with large, electron lucent vesicles are seen. These clear granules vary from 1150–1750 Å in diameter and often exhibit broken limiting membranes. Few small vesicles are seen near the plasma membrane of these endings; however, instances of invaginations of the plasma membrane occur. The significance of endings filled with clear granules is discussed.Supported by a grant from the National Research Council of Canada (No. A-4675).     

The dopamine D2 receptor: two molecular forms generated by alternative splicing.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.     

Myeloid dendritic cells from B6.NZM Sle1/Sle2/Sle3 lupus-prone mice express an IFN signature that precedes disease onset

Patients with systemic lupus erythematosus show an overexpression of type I IFN-responsive genes that is referred to as "IFN signature." We found that B6.NZMSle1/Sle2/Sle3 (Sle1,2,3) lupus-prone mice also express an IFN signature compared with non-autoimmune C57BL/6 mice. In vitro, myeloid dendritic cells (mDCs) (GM-CSF bone marrow-derived dendritic cells; BMDCs) from Sle1,2,3 mice constitutively overexpressed IFN-responsive genes such as IFN-β, Oas-3, Mx-1, ISG-15, and CXCL10 and members of the IFN signaling pathway STAT1, STAT2, and IRF7. The IFN signature was similar in Sle1,2,3 BMDCs from young, pre-autoimmune mice and from mice with high titers of autoantibodies, suggesting that the IFN signature in mDCs precedes disease onset and is independent from the autoantibodies. Sle1,2,3 BMDCs hyperresponded to stimulation with IFN-α and the TLR7 and TLR9 agonists R848 and CpGs. We propose that this hyperresponse is induced by the IFN signature and only partially contributes to the signature, as oligonucleotides inhibitory for TLR7 and TLR9 only partially suppressed the constitutive IFN signature, and pre-exposure to IFN-α induced the same hyperresponse in wild-type BMDCs as in Sle1,2,3 BMDCs. In vivo, mDCs and to a lesser extent T and B cells from young prediseased Sle1,2,3 mice also expressed the IFN signature, although they lacked the strength that BMDCs showed in vitro. Sle1,2,3 plasmacytoid DCs expressed the IFN signature in vitro but not in vivo, suggesting that mDCs may be more relevant before disease onset. We propose that Sle1,2,3 mice are useful tools to study the role of the IFN signature in lupus pathogenesis.     

Functional and structural characterization of the integrase from the prototype foamy virus

Establishment of the stable provirus is an essential step in retroviral replication, orchestrated by integrase (IN), a virus-derived enzyme. Until now, available structural information was limited to the INs of human immunodeficiency virus type 1 (HIV-1), avian sarcoma virus (ASV) and their close orthologs from the Lentivirus and Alpharetrovirus genera. Here, we characterized the in vitro activity of the prototype foamy virus (PFV) IN from the Spumavirus genus and determined the three-dimensional structure of its catalytic core domain (CCD). Recombinant PFV IN displayed robust and almost exclusively concerted integration activity in vitro utilizing donor DNA substrates as short as 16 bp, underscoring its significance as a model for detailed structural studies. Comparison of the HIV-1, ASV and PFV CCD structures highlighted both conserved as well as unique structural features such as organization of the active site and the putative host factor binding face. Despite possessing very limited sequence identity to its HIV counterpart, PFV IN was sensitive to HIV IN strand transfer inhibitors, suggesting that this class of inhibitors target the most conserved features of retroviral IN-DNA complexes.