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Receptors encoded by the natural killer (NK) cell gene complex (such as NKG2D) govern the reactivity of NK cells. However, the function and ligand(s) of the NK cell gene complex-encoded human NK cell receptor NKp80 remain elusive. Here we demonstrate that NKp80 binds to the genetically linked 'orphan' receptor AICL, which, like NKp80, is absent from rodents. We defined AICL as a myeloid-specific activating receptor that is upregulated by Toll-like receptor stimulation. AICL-NKp80 interactions promoted NK cell-mediated cytolysis of malignant myeloid cells. In addition, during crosstalk between NK cells and monocytes, NKp80 stimulated the release of proinflammatory cytokines from both cell types. Thus, by specifically bridging NK cells and myeloid cells, NKp80-AICL interactions may contribute to the initiation and maintenance of immune responses at sites of inflammation.

The interaction of lipopolysaccharide with CD14 plays a key role in signaling that activates an early defense against pathogens but also contributes to the development of sepsis and septic shock. Here we have mapped the entire 356-amino-acid protein with synthetic 20-amino-acid peptides and have identified a new lipopolysaccharide-binding domain with a strong LPS-neutralizing activity. Moreover, analysis of the structure-activity relationship of this peptide, which corresponds to amino acids 81-100 of human CD14, revealed that leucines 87, 91, and 94 are essential for these activities. The functional relevance of these residues was confirmed by cellular expression of mutant CD14 proteins that are no longer able to bind LPS. Furthermore, the peptide provided a basis for the generation of highly soluble analogues with stronger lipopolysaccharide-neutralizing activity.

Cationic cell-penetrating peptides (CPPs) have been used widely as delivery vectors for the import of molecules that otherwise do not cross the plasma membrane of eukaryotic cells. In this work, we demonstrate that the three cationic CPPs, Antennapedia homeodomain-derived peptide (Antp), nona-arginine and Tat-derived peptide, inhibit tumour necrosis factor (TNF)-mediated signal transduction. This inhibition is based on the downregulation of TNF receptors at the cell surface by induction of internalization. In contrast to TNF-dependent receptor internalization, no receptor activation occurs. The receptor downregulation is not restricted to the CPPs. Remarkably, the HIV-1 Tat protein itself also induces the internalization of TNF receptors. The dynamin dependence of the internalization, as well as the fact that epidermal growth factor receptors are also internalized, suggest a general induction of clathrin-dependent endocytosis as the mechanism of action. The significance of these findings for the use of cationic CPPs in the import of bioactive peptides is demonstrated here using a conjugate consisting of Antp and a Smac protein-derived cargo peptide. The cargo alone, when introduced into cells by electroporation, enhanced TNF-induced apoptosis by inhibiting the anti-apoptotic action of IAPs (inhibitor of apoptosis proteins). For the Antp-Smac conjugate at concentrations below 40 muM the inhibitory effect of the Antp peptide compensated for the pro-apoptotic activity of the cargo, and led to the protection of cells against TNF-mediated apoptosis. These data provide important new information for the use of cationic CPPs for the cellular delivery of bioactive molecules.

The fluorogenic substrate 6,8-difluoro-4-methylumbiliferyl phosphate (DIFMUP) has been widely used for the detection of serine and threonine phosphatase activities. Here we describe the use of this substrate for the characterization of protein tyrosine phosphatases (PTPs) and for the screening for PTP inhibitors. The measured kinetic and inhibitor constants for DIFMUP cleavage were comparable with those of the widely used but less discriminative and practicable substrates, para-nitrophenylphosphate and phosphotyrosine-containing peptides, respectively. Furthermore, the continuous and highly sensitive assay allows fast and accurate investigations of the type, kinetic behavior, and binding mode of small-molecule inhibitors. We discuss the validation of this assay system for various PTPs and its use in inhibitor screening for PTP1B.

Accessory/co-stimulatory molecules on the surface of T cells are capable of regulating activation signals. Two of these, CD5 and CD6, are molecules from the scavenger receptor cysteine rich (SRCR) superfamily. Partial sequences for the ligands of these molecules, known as CD72 and CD166 (or ALCAM), respectively, are provided for Bos taurus in this communication. Using highly conserved regions between the corresponding human and mouse genes, primers were designed and reverse transcription polymerase chain reaction was used to generate cDNA from bovine PBMC RNA. cDNA clones of several hundred base pairs in length were created and sequenced. The results showed 81% homology between bovine and human CD72 nucleotide sequences and 93% homology for the CD166 sequences. Similar levels of homology are seen between the corresponding human and mouse cDNA sequences.

Bovine gammadelta T cells are stimulated to proliferate by autologous monocytes. This is referred to as the autologous mixed leucocyte reaction (AMLR). It has been shown previously that the stimulatory component is constitutively expressed on the monocyte plasma membrane and is a protein or has a protein moiety. Here we showed that gammadelta T-cell responses to the monocytes requires interaction with the T-cell receptor because Fab1 fragments of a monoclonal antibody (mAb) that reacts with the delta chain of the T-cell receptor blocked proliferation in the AMLR. Monocyte molecules involved in stimulation were also characterized further by biochemical and immunological methods. A mAb, named M5, was generated by immunizing mice with bovine monocytes and shown to block the ability of monocytes to stimulate in the AMLR. Treatment of monocytes or monocyte membranes with high salt, chelating agents or phospholipase C did not affect their ability to stimulate gammadelta T-cell proliferation or reactivity with mAb M5 indicating the ability of monocytes to stimulate does not involve peripheral membrane components or a glycosyl-phosphatidylinsositol (GPI)-anchored components. Hence it was concluded that the stimulation occurred as a result of intergral membrane proteins including that recognized by mAb M5. The ligand for mAb M5 was on all bovine monocytes and to a lower level on granulocytes but not on lymphocytes. MAb M5 also reacted with sheep monocytes but not with human monocytes or murine macrophages, in agreement with a previous reports that sheep monocytes but not human or mouse mononuclear phagocytes have the capacity to stimulate bovine gammadelta T cells in in vitro cultures. The level of expression of the M5 ligand was not altered by gamma-irradiation or culture of monocytes with lipopolysaccharide but it was decreased following culture with interferon-gamma-containing cell culture supernatants.

The insulin signal transduction cascade provides a number of sites downstream of the insulin receptor (IR) for cross-talk from other signaling pathways. Tyrosine phosphorylation of the IR substrates IRS-1/2 and metabolic insulin-mimetic activity in insulin-responsive cells can be provoked by soluble phosphoinositolglycans (PIG), which trigger redistribution from detergent-insoluble glycolipid-enriched raft domains (DIGs) to other areas of the plasma membrane and thereby activation of nonreceptor tyrosine kinases (NRTK) [Müller, G., Jung, C., Wied, S., Welte, S., Jordan, H., and Frick, W. (2001) Mol. Cell. Biol. 21, 4553-4567]. Here we describe that stimulation of glucose transport in isolated rat adipocytes by a different stimulus, the sulfonylurea glimepiride, is also based on IRS-1/2 tyrosine phosphorylation and downstream insulin-mimetic signaling involving activation of the NRTK, pp59(Lyn), and pp125(Fak), as well as tyrosine phosphoryation of the DIGs component caveolin. As is the case for PIG 41, glimepiride causes the concentration-dependent dissociation of pp59(Lyn) from caveolin and release of this NRTK and the glycosyl-phosphatidylinositol-anchored (GPI) proteins, Gce1 and 5'-nucleotidase, from total and anti-caveolin-immunoisolated DIGs. This results in their movement to detergent-insoluble raft domains of higher buoyant density (non-DIGs areas). IRS-1/2 tyrosine phosphorylation and glucose transport activation by both glimepiride and PIG are blocked by introduction into adipocytes of the caveolin scaffolding domain peptide which mimicks the negative effect of caveolin on pp59(Lyn) activity. Tyrosine phosphorylation of the NRTK, IRS-1/2, and caveolin as well as release of the NRTK and GPI proteins from DIGs and their redistribution into non-DIGs areas in response to PIG is also inhibited by treatment of intact adipocytes with either trypsin plus salt or N-ethylmaleimide (NEM). In contrast, the putative trypsin/salt/NEM-sensitive cell surface component (CIR) is not required for glimepiride-induced glucose transport, IRS-1/2 tyrosine phosphorylation, and redistribution of GPI proteins and NRTK. The data suggest that CIR is involved in concentrating signaling molecules at DIGs vs detergent-insoluble non-DIGs areas. These inhibitory interactions are relieved in response to putative physiological (PIG) or pharmacological (sulfonylurea) stimuli via different molecular mechanisms (dependent on or independent of CIR, respectively) thereby inducing IR-independent positive cross-talk to metabolic insulin signaling.

Caveolae and caveolin-containing detergent-insoluble glycolipid-enriched rafts (DIG) have been implicated to function as plasma membrane microcompartments or domains for the preassembly of signaling complexes, keeping them in the basal inactive state. So far, only limited in vivo evidence is available for the regulation of the interaction between caveolae-DIG and signaling components in response to extracellular stimuli. Here, we demonstrate that in isolated rat adipocytes, synthetic intracellular caveolin binding domain (CBD) peptide derived from caveolin-associated pp59(Lyn) (10 to 100 microM) or exogenous phosphoinositolglycan derived from glycosyl-phosphatidylinositol (GPI) membrane protein anchor (PIG; 1 to 10 microM) triggers the concentration-dependent release of caveolar components and the GPI-anchored protein Gce1, as well as the nonreceptor tyrosine kinases pp59(Lyn) and pp125(Fak), from interaction with caveolin (up to 45 to 85%). This dissociation, which parallels redistribution of the components from DIG to non-DIG areas of the adipocyte plasma membrane (up to 30 to 75%), is accompanied by tyrosine phosphorylation and activation of pp59(Lyn) and pp125(Fak) (up to 8- and 11-fold) but not of the insulin receptor. This correlates well to increased tyrosine phosphorylation of caveolin and the insulin receptor substrate protein 1 (up to 6- and 15-fold), as well as elevated phosphatidylinositol-3' kinase activity and glucose transport (to up to 7- and 13-fold). Insulin-mimetic signaling by both CBD peptide and PIG as well as redistribution induced by CBD peptide, but not by PIG, was blocked by synthetic intracellular caveolin scaffolding domain (CSD) peptide. These data suggest that in adipocytes a subset of signaling components is concentrated at caveolae-DIG via the interaction between their CBD and the CSD of caveolin. These inhibitory interactions are relieved by PIG. Thus, caveolae-DIG may operate as signalosomes for insulin-independent positive cross talk to metabolic insulin signaling downstream of the insulin receptor based on redistribution and accompanying activation of nonreceptor tyrosine kinases.

The nematode Eustrongylides ignotus was found in peritoneal lesions of several great blue herons (Ardea herodias) submitted for necropsy from a wildlife rehabilitation center in northern Delaware. Prior to death, signs of disease included ataxia, emaciation, weakness, and anemia. Blood collection was not uniformly performed, but in cases where it was performed, affected birds demonstrated abnormal clinical hematology. Postmortem findings included numerous lesions associated with verminous peritonitis. Significant histologic granulomatous response to the presence of these organisms was noted, particularly in the proventricular specimens. Other organs involved included intestine, spleen, pancreas, and liver.

Aim: The aim of the study was to determine the absolute bioavailability and pharmacokinetics after a single dose oral administration in comparison to i.v. administration of 14C-labelled distigmine-bromide (14C-Ubretid) in healthy male volunteers., Results: After the intravenous administration, distigmine is eliminated from the body by renal excretion (85%), and for a small fraction by biliary excretion in the feces (4%). This situation is reversed after an oral administration, where 6.5% of the dose is recovered from the urine and 88% from the feces. This means that distigmine after oral administration is hardly absorbed, the calculated bioavailability is 4.65%., Conclusion: The mean absorption time (MAT) after oral administration was 10 h, influencing the t(1/2alpha) (1.4 vs 4.5 h) and the t(1/2beta) (60 vs 70 h) to higher values than after the i.v. administration (p < 0.05).

Clinical phase I pharmacokinetic studies with lornoxicam were performed with the 4 mg dose of lornoxicam. Lornoxicam was administered as an aqueous solution both orally and intravenously to young, healthy, male volunteers. The total excretion of lornoxicam via urine and faeces after oral administration was determined by administering 14C-labelled compound. The results show that the parent compound and the main metabolite, 5'-hydroxy-lornoxicam, were found in plasma. However, in urine, no lornoxicam was detected, only 5'-hydroxy-lornoxicam. After oral as well as intravenous administration, a short terminal half-life of lornoxicam in the range of 4-5 hours was found. Given orally as solution, lornoxicam was rapidly and almost completely absorbed.

Antacids are used in the treatment of upper gastrointestinal side-effects during therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). Since pharmacokinetic interactions between antacids and NSAIDs have been reported, it was investigated whether aluminium and magnesium hydroxide (Maalox as oral suspension) or aluminium hydroxide and calcium carbonate (Solugastril as oral gel) influenced the bioavailability of Lornoxicam (rINN), a new potent NSAID from the chemical group of the oxicams. Eighteen male volunteers were given 4 mg of Lornoxicam as a film-coated tablet either alone or together with 10 ml of Maalox or 10 g of Solugastril in an open, randomized, three-way cross-over study. The levels of Lornoxicam in plasma were determined by HPLC following solid-phase extraction. It was found that none of the antacids changed significantly any of the following pharmacokinetic parameters: elimination half-life (t1/2 beta), concentration at peak time (Cmax), time to reach the peak (tmax) and area under the curve to infinity (AUCo-infinity). The results indicate that the concomitant administration of antacids did not influence the pharmacokinetic profile of Lornoxicam. Furthermore they confirm the short elimination half-life of Lornoxicam in man, which is markedly shorter than that of other oxicam-type compounds.

Lornoxicam is a new, highly potent antirheumatic agent which is an oxicam derivative. Although highly potent as a cyclo-oxygenase inhibitor, the compound does not cause inhibition of 5-lipoxygenase and does not appear to shunt arachidonic acid through this cascade. This powerful inhibition of cyclo-oxygenase has manifested itself as highly potent analgesic and anti-inflammatory effects in animal studies and also prevented the bone destruction which normally occurs in the adjuvant polyarthritic rat. To explain this finding, studies have demonstrated that lornoxicam inhibits polymorphonuclear (PMN)-leukocyte migration; inhibits the release of superoxide from human PMN-leukocytes; inhibits the release of platelet derived growth factor (PDGF) from human platelets and stimulates the synthesis of proteoglycans in cartilage in tissue culture. Lornoxicam is well absorbed and has a plasma t1/2 in man of 4 hours which is distinctly different from other oxicams. It is metabolized in animals and in man to the 5'-hydroxy-metabolite which is inactive in pharmacological tests. In vitro and in vivo animal safety studies have demonstrated both subchronically and chronically that lornoxicam manifests no unusual toxicity, is not a mutagen nor is it tumorigenic and causes no fetal teratogenicity in reproduction studies.

Of 105 juvenile American robins (Turdus migratorius L.) examined for fecal parasites, 77.1% were infected with one or more species of endoparasite. Syngamus sp. was the most commonly encountered parasite, found in 57.1% of the birds. There was a significant association between the presence of Syngamus sp. eggs in feces and signs of respiratory-tract disease. A single oral dose of fenbendazole (100 mg/kg of body weight) eliminated Syngamus sp. infection from all of 18 birds treated, yet 10 of 16 untreated controls apparently were "self-cured" over the 12-day period of observation.

The pharmacokinetics of midodrin (alpha-2,5-dimethoxyphenyl-beta-glycinamidoethanol hydrochloride, ST 1085) and its main metabolite ST 1059 (alpha-2,5-dimethoxyphenyl-beta-aminoethanol hydrochloride) have been investigated in 12 male healthy volunteers. 2.5 mg midodrin hydrochloride were applied intravenously, as drinking solution or as tablet (Gutron) according to a randomized cross-over design. Plasma and urine samples collected up to 24 h after application were analyzed by high-performance liquid chromatography with fluorescence detection. The mean maximum concentration in plasma for midodrin was ca. 10 ng/ml 20-30 min after oral administration, for ST 1059 ca. 5 ng/ml after 1 h. Midodrin was eliminated with a terminal half-life of 0.5 h. The half-life of ST 1059 was determined to be 3 h. The mean area under the plasma-level vs. time curve (AUC) of ST 1059 after administration of 2.5 mg midodrin i.v. was 28.7 ng X h/ml, and as drinking solution or as tablet 25.7 and 25.6 ng X h/ml, respectively. The data of 10 volunteers could be used for the calculations of the bioavailability of ST 1059 by the AUC. Assuming an interval of equivalence of 0.75-1.25 because of the relatively small number of volunteers, the three galenical formulations are considered to be equivalent.