Bovine glomerular basement membrane. Location and structure of the asparagine-linked oligosaccharide units and their potential role in the assembly of the 7 S collagen IV tetramer

Collagen IV contains an amino-terminal tetramerization domain (7 S) that is involved in aggregation and cross-linking as part of the process of self-assembly of the collagen IV matrix of basement membranes. We determined the structure and location of the Asn-linked oligosaccharides of the 7 S tetramer. Two glycopeptides, GP-1 and GP-2, were isolated from tryptic digests of the 7 S tetramer and were characterized. GP-1 and GP-2 are derived from the alpha 1(IV) chain and the alpha 2(IV) chain, respectively. Each glycopeptide contained one sequence, -Asn-Xaa-Thr-, which was shown to be N-glycosylated at Asn, corresponding to position 126 of the alpha 1 chains and 138 of the alpha 2 chain. 1H NMR spectroscopic analysis of the oligosaccharide is a biantennary N-acetyllactosamine type of N-linked oligosaccharide with a broad heterogeneity in the presence of the sugar residues at their nonreducing termini as indicated. [formula: see text] The location of the Asn-linked oligosaccharide units and Hyl-linked disaccharide units and their orientation with respect to the surface of the triple helix were calculated using two models. We conclude that both units are important determinants in the assembly of the 7 S tetramer.     

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.     

Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes

The tissue localization or "homing" of circulating lymphocytes is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. In peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches and appendix), and sites of chronic inflammation, for example, lymphocytes leave the blood by adhering to and migrating between those endothelial cells lining postcapillary high endothelial venules (HEV). Functional analyses of lymphocyte interactions with HEV have shown the lymphocytes can discriminate between HEV in different tissues, indicating that HEV express tissue-specific determinants or address signals for lymphocyte recognition. We recently described such a tissue-specific "vascular addressin" that is selectively expressed by endothelial cells supporting lymphocyte extravasation into mucosal tissues and that appears to be required for mucosa-specific lymphocyte homing (Streeter, P. R., E. L. Berg, B. N. Rouse, R. F. Bargatze, and E. C. Butcher. 1988. Nature (Lond.). 331:41-46). Here we document the existence and tissue-specific distribution of a distinct HEV differentiation antigen. Defined by monoclonal antibody MECA-79, this antigen is expressed at high levels on the lumenal surface and in the cytoplasm of HEV in peripheral lymph nodes. By contrast, although MECA-79 stains many HEV in the mucosal Peyer's patches, expression in most cases is restricted to the perivascular or ablumenal aspect of these venules. In the small intestine lamina propria, a mucosa-associated site that supports the extravasation of lymphocytes, venules do not stain with MECA-79. Finally, we demonstrate that MECA-79 blocks binding of both normal lymphocytes and a peripheral lymph node-specific lymphoma to peripheral lymph node HEV in vitro and that it also inhibits normal lymphocyte homing to peripheral lymph nodes in vivo without significantly influencing lymphocyte interactions with Peyer's patch HEV in vitro or in vivo. Thus, MECA-79 defines a novel vascular addressin involved in directing lymphocyte homing to peripheral lymph nodes.     

The histochemical specificity of High Iron Diamine—Alcian Blue

Summary The specificity of the High Iron Diamine—Alcian Blue pH2.5 (HID—AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine—Alcian Blue pH 1.0 demonstrated that complete or almost complete staining ofO-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID—AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied transitional mucosa in human colonic diseases. Caution should be used in drawing conclusions from the use of HID—AB 2.5 without confirmatory evidence from other more specific procedures.     

Neurotensin regulation of macrophage colony-stimulating factor-stimulated in vitro myelopoiesis

Neurotensin, at less than or equal to 10(-9) M, in the presence of an optimal concentration of macrophage CSF (CSF-1), stimulated a dose-dependent enhancement of colony formation by murine marrow-derived mononuclear phagocyte progenitor cells. The additional colonies arose from the cell cycle and Ia Ag-positive subpopulation previously identified as two-signal-dependent progenitors. Two-signal colony formation diminished when the peptide was added at concentrations greater than 10(-9) M. Neurotensin binds specifically to two distinct receptors, a high affinity receptor (KD approximately 10(-9) M) and a lower affinity (KD approximately 10(-7) M) receptor identified as the tuftsin receptor. Rat liver ferritin and an inhibitory tuftsin analog. (ALA1)-tuftsin, which inhibit two-signal colony formation stimulated by tuftsin and tuftsin-like peptides in combination with CSF-1, did not inhibit colony formation stimulated by CSF-1 and 10(-9) M neurotensin. Both inhibitors, however, reversed the loss of two-signal colony growth in the presence of higher neurotensin concentrations. Neurotensin fragment 1-6, unlike ferritin and (ALA1)-tuftsin, inhibited two-signal colony formation stimulated by 10(-9) M neurotensin. However, like ferritin and (ALA1)-tuftsin, fragment 1-6 permitted full expression of two-signal colony formation in the presence of CSF-1 and 10(-7) M neurotensin. The data indicated that occupancy of both receptors at neurotensin concentrations greater than 10(-9) M might be responsible for the diminished progenitor response. The data further support a potential role for neurotensin as an inflammatory mediator. In addition to direct effects on mature phagocytic leukocytes, neurotensin, at least in vitro can influence the production of new mononuclear phagocytes.     

Is Diurodrilus an annelid?

Interstitial marine meiofaunal worms of the genus Diurodrilus have always been considered part of Annelida, either as basal or derived, though generally with reference to Dinophilidae. New evidence shows that Diurodrilus has a unique anatomy, and lacks key annelid features, possibly even segmentation. We assessed the systematic position of Diurodrilus among other protostome animals via light microscopy, confocal laser scanning microscopy, and transmission electron microscopy studies of anatomy, focusing on musculature, the nervous system, as well as molecular sequence data. We show that there is little morphological or molecular evidence to support a relationship with Dinophilidae or any other annelids. Diurodrilus has some similarities to Micrognathozoa, though the latter shows complex jaws. On the basis of the configuration of the nervous system and the cuticle we regard Diurodrilus to belong to Spiralia, possibly close to Annelida; however, until further evidence is acquired it should be regarded as incertae sedis in this large animal clade. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Live fast,die young: the life cycle of the brooding feather star Aporometra wilsoni (Echinodermata: Crinoidea)

We present here the first documentation of the entire life cycle of a crinoid. A population of the diminutive feather star Aporometra wilsoni, which broods larvae, was sampled near Adelaide, in the Gulf St Vincent, South Australia, every fortnight between February 2004 and February 2005. Body size, sex, and reproductive status were recorded for 30–50 individuals collected on each occasion. The population showed a sex ratio of 1:1, with unequivocal male and female specimens found in 10 of 13 months. The average size (arm length) of individuals increased until June, when it stabilized and females began to brood larvae. Females were found brooding larvae until November, when adults began senescing. By January, the majority of the population consisted of small recruits. The entire life cycle of this small ovoviviparous crinoid occurs over a single year, a life cycle that is unique among echinoderms.     

Phenotypic and functional studies on ocular T cells during herpetic infections of the eye.

Herpetic stromal keratitis an inflammatory disease of the eye resulting from herpes simplex virus type 1 infection, is a common cause of blindness. The disease is generally considered to represent an immunopathologic response, but the exact mechanism remains in doubt and is subject to debate. We have investigated the nature of inflammatory cells in the eye and have isolated ocular cells to establish their phenotype and determine some of their functions. By means of immunocytochemistry and cytofluorography, the only T lymphocyte subset detectable at any stage of infection of BALB/c mice were CD4+ cells. However CD8+ T cells were readily detectable in draining lymph nodes (DLN). Assays for cells with HSV-1-specific cytotoxic function of both CD4+ class II restricted and CD8+ class I-restricted activity were performed. Although in DLN both cell types were found, among ocular cells only CD4+ cytotoxic cells were evident. The frequencies of CD4+ CTL-precursor in eyes were determined and found to be at least 8- to 10-fold less than found in DLN. The number of CTL-precursor in an individual eye was estimated to be 20 or less. Our results further support the notion that CD4+ mediate the immunopathology of herpetic stromal keratitis, but on quantitative grounds cast doubt on the idea that cytotoxicity is the principal mechanism involved.     

Three Loci Modify Growth of a Transgene-Induced Mammary Tumor: Suppression of Proliferation Associated with Decreased Microvessel Density

In earlier studies it was observed that the genetic background significantly affected the phenotype of a transgene-induced mammary tumor. Tumors arising in an (I/LnJ x PyMT) F1 hybrid background appeared earlier than in the FVB/N-TgN(MMTV-PyVT)(634Mul) parent, but accumulated less tumor mass, indicating a net decrease in tumor growth. Quantitative genetic mapping in a backcross identified three loci that were associated with the decreased proliferative capacity of the I/LnJ F1 tumors. Molecular analysis of the tumors suggests that these loci may act by restricting the tumor's ability to recruit microvessels. The three loci, designated Mmtg1-3, are unlinked to the angiogenic genes Fgf2, Flt1, Flk4, Flk1, Vegf, and Vegfc, as well as the precursors of the endogenous antiangiogenic molecules angiostatin and endostatin. The Mmtg loci may therefore provide novel targets for antiangiogenic therapeutic strategies.