Adhesion and Endocytosis of Calcium Oxalate Crystals on Renal Tubular Cells

The present investigation was designed to study interactions between Madin-Darby canine kidney (MOCK) cells and calcium oxalate monohydrate (COM) crystals and to clarify the significance of these crystal-cell interactions in stone pathogenesis. MOCK cells cultured in the presence of COM crystals showed a time-dependent uptake of crystals; this was specific for COM crystals. In the dynamic model system designed to study these phenomena under more physiological conditions, COM crystals adhered to the cell surface and were subsequently internalized. In this endocytotic process, the microvilli of the cell appeared to play an important role. The observation by scanning electron microscopy of complexes consisting of aggregated COM crystals and cell debris led us to speculate that adhesion and endocytosis of crystals might provide the calculus nidus for aggregation and retention of crystals in the renal tubule. Furthermore, glycosaminoglycans and the macromolecular fraction of human urine were shown to have the ability to inhibit the cellular uptake of crystals. Evidence that similar processes may also occur in vivo was obtained using an experimental stone model in rats. Our experiments revealed that most of the COM crystals adhered to the tubular cells and some crystals were endocytosed by the cell. Thus, these crystal-cell interactions might be one of the earliest processes in the formation of kidney stones. Further elucidation of the mechanism and the regulatory factors involved in this process may provide new insight into stone pathogenesis

Reproductive site selection and characteristics of sources and sinks in an Italian tree frog metapopulation (Hyla intermedia, Boulenger 1882)

Ce travail est une des premières études extensives de terrain sur la sélection des lieux de reproduction et sur le succès reproductif d'une vaste population de Rainette italienne (Hyla intermedia), un endémique de l'Italie, qui n'a été reconnu que récemment comme distinct de la Rainette verte Européenne (Hyla arborea). Un inventaire précis a été conduit en 1997 et 1998 dans tous les points d'eau du territoire de la Réserve Naturelle de la « Baraggia del Piano Rosa » (Piémont, Italie du Nord). Ce milieu particulier (lande de bruyère continentale du piémont des Alpes) d'origine anthropique est caractérisé par un paysage ouvert hétérogène, avec de nombreuses petites zones humides saisonnières. Nous avons observé une bonne présence de Rainettes italiennes, dont la distribution spatiale montrait une structure métapopulationnelle typique. Les données sur les paramètres environnementaux caractéristiques des sites et de leurs alentours ont été traitées par analyse discriminante, en fonction de la présence et de l'utilisation pour la reproduction de H. intermedia et de leur rôle dans un modèle de métapopulation « source-puits ». Ces analyses montrent que les rainettes éviteraient presque complètement les points d'eau permanente profonds et ombragés et suggèrent une thermophilie marquée. Le dérangement générique causé par les activités humaines paraît influencer négativement la reproduction, mais pas la présence d'H. intermedia. La préférence pour les stages intermédiaires et avancés de la succession hydrosérale a aussi été mise en évidence. Notre étude a indiqué l'importance de certains sites « sources », caractérisés par un «trade-off» positif entre température et stabilité de l'eau pour l'accomplissement de la cruciale phase larvaire.In this work we carried out one of the first extensive field researches on the reproductive site selection and the reproductive success of a wide metapopulation of Italian Tree Frog (Hyla intermedia), an Italian endemism that only recently has been recognized as different from the better known European Tree Frog (Hyla arborea). An accurate census of al! the water sites in the territory of the "Baraggia del Piano Rosa" Natural Reserve (Piedmont, Northem ltaly) was conducted in the years 1 997 and 1998. This particular environment (anthropogenic sub-montane continental heathland) is characterized by a heterogeneous open landscape and many small seasonal wet zones. We observed a good overall presence of tree frogs and their spatial distribution showed a typical metapopulation structure. Data collected on many environmental parameters characterizing the sites and their surroundings were investigated using Discriminant Function Analysis, in function of the presence and reproductive use by H. intermedia and their role in a "source-sink" metapopulation model. These analyses show that tree frogs avoided almost completely deep, shadowed, permanent water sites, suggesting the strong thermophily of the species. Generic disturbance caused by human activities resulted to influence negatively H. intermedia reproduction, but not their presence. The preference for intermediate and advanced phases in the hydroseral succession was also evidenced. Our study indicated the importance of some "source" sites characterized by a trade-off between water temperature and stability for the successful completion of the critical larval stage

Organizer-Like Reticular Stromal Cell Layer Common to Adult Secondary Lymphoid Organs

Abstract Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1+ICAM-1+MAdCAM-1+ reticular cell line that can produce CXCL13 upon LTβR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2−/− mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs

Development of caged non-hydrolyzable phosphoamino acids and application to photo-control of binding affinity of phosphopeptide mimetic to phosphopeptide-recognizing protein

The design and synthesis of caged non-hydrolyzable phospho-serine, -threonine, and -tyrosine derivatives that generate parent non-hydrolyzable phosphoamino acids, containing a difluoromethylene unit instead of the oxygen of a phosphoester, after UV-irradiation are described. The caged non-hydrolyzable amino acids were incorporated into peptides by standard Fmoc solid-phase peptide synthesis, and the obtained peptides were successfully converted to the parent non-hydrolyzable phosphopeptides by UV-irradiation. Application of the caged non-hydrolyzable phosphoserine-containing peptide to photo-control the binding affinity of the peptide to 14-3-3β protein is also reported

Nepmucin, a novel HEV sialomucin, mediates L-selectin–dependent lymphocyte rolling and promotes lymphocyte adhesion under flow

Lymphocyte trafficking to lymph nodes (LNs) is initiated by the interaction between lymphocyte L-selectin and certain sialomucins, collectively termed peripheral node addressin (PNAd), carrying specific carbohydrates expressed by LN high endothelial venules (HEVs). Here, we identified a novel HEV-associated sialomucin, nepmucin (mucin not expressed in Peyer's patches [PPs]), that is expressed in LN HEVs but not detectable in PP HEVs at the protein level. Unlike conventional sialomucins, nepmucin contains a single V-type immunoglobulin (Ig) domain and a mucin-like domain. Using materials affinity-purified from LN lysates with soluble L-selectin, we found that two higher molecular weight species of nepmucin (75 and 95 kD) were decorated with oligosaccharides that bind L-selectin as well as an HEV-specific MECA-79 monoclonal antibody. Electron microscopic analysis showed that nepmucin accumulates in the extended luminal microvillus processes of LN HEVs. Upon appropriate glycosylation, nepmucin supported lymphocyte rolling via its mucin-like domain under physiological flow conditions. Furthermore, unlike most other sialomucins, nepmucin bound lymphocytes via its Ig domain, apparently independently of lymphocyte function–associated antigen 1 and very late antigen 4, and promoted shear-resistant lymphocyte binding in combination with intercellular adhesion molecule 1. Collectively, these results suggest that nepmucin may serve as a dual-functioning PNAd in LN HEVs, mediating both lymphocyte rolling and binding via different functional domains

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes.

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5\u27-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response

Development of UV-responsive catch-and-release system of a cysteine protease model peptide

Cysteine proteases are attractive drug targets due to their involvement in a wide variety of diseases. To evaluate the potential of a particular protease as a drug target, use of a reagent that controls activity of the protease is indispensable. In this context, we have developed a catch-and-release reagent that first forms a covalent bond with the active center thiol of a cysteine protease to suppress its activity and then is removed by UV-irradiation to release the parent active protease. In this paper, the design and synthesis of a catch-and-release reagent of thiols are described. Its application to caging (catch) and UV-induced uncaging (release) of a model peptide derived from an active site of caspase-9 and introduction of a recognition moiety on the reagent are also reported

Development of a Reduction‐Responsive Amino Acid that Induces Peptide Bond Cleavage in Hypoxic Cells

Utilization of a hypoxia-responsive amino acid is indispensable in the preparation of hypoxic tumor-specific peptidyl prodrugs. Bioreduction of a nitro group is among the most attractive triggering reactions in the hypoxia-responsive prodrugs. In this paper, design and synthesis of a reduction-responsive amino acid that induces peptide bond cleavage after reduction of the nitro group are described. Application to hypoxia-responsive peptide bond cleavage system is also reported

Development of a Reduction‐Responsive Amino Acid that Induces Peptide Bond Cleavage in Hypoxic Cells

Utilization of a hypoxia-responsive amino acid is indispensable in the preparation of hypoxic tumor-specific peptidyl prodrugs. Bioreduction of a nitro group is among the most attractive triggering reactions in the hypoxia-responsive prodrugs. In this paper, design and synthesis of a reduction-responsive amino acid that induces peptide bond cleavage after reduction of the nitro group are described. Application to hypoxia-responsive peptide bond cleavage system is also reported