Requirement of cysteine-rich repeats of the Fas receptor for binding by the Fas ligand

The Fas receptor is a member of a family of cell death receptors, including tumor necrosis factor receptor I (TNFR I), death receptor 3 and 4 (DR3 and DR4), and cytopathic avian receptor 1 (CAR1). The Fas receptor is composed of several discrete domains, including three cysteine-rich domains (CRDs), a transmembrane domain, and an intracellular domain responsible for transmitting an apoptotic signal. While the mechanism of Fas-mediated cell death has become elucidated, the requirements for Fas ligand binding to the receptor have not been fully defined. Using a series of chimeric Fc-receptor fusion proteins between the human Fas receptor and TNFR I, each cysteine-rich domain of Fas was found to be required for interaction with the Fas ligand. Interestingly, TNFR I CRD1 could partially substitute for the Fas CRD1. The importance of this domain was underscored by the analysis of a Fas extracellular mutation (C66R), which resulted in a complete loss of ligand binding. This mutation was cloned from a human patient suffering from Canale-Smith syndrome, which is characterized by autoimmunity resembling that observed in the lpr and lprcg mice. The localization of essential ligand binding domains in the Fas receptor correlated exactly with the ability of the Fas receptor fusion proteins to prevent cell death mediated by the Fas ligand.     

Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.     

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

RANTES-induced T cell activation correlates with CD3 expression

The chemokine RANTES induces a unique biphasic cytoplasmic Ca2+ signal in T cells. The first phase of this signal, similar to that of other chemokines, is G-protein mediated and chemotaxis associated. The second phase of this signal, unique to RANTES and evident at concentrations greater than 100 nM, is tyrosine kinase linked and results in a spectrum of responses similar to those seen with antigenic stimulation of T cells. We show here that certain jurkat T cells responded to RANTES solely through this latter pathway. A direct correlation between the RANTES-induced second phase response and CD3 expression was demonstrated in these cells. Sorting the Jurkat cells into CD3(high) and CD3(low) populations revealed that only the CD3(high) cells were responsive to RANTES. Furthermore, stimulation of these Jurkat cells with anti-CD3 mAb significantly depresses their subsequent response to RANTES. While a RANTES-specific chemokine receptor is expressed at a low level on these Jurkat cells, the RANTES-induced activation is dependent on the presence of the TCR. Thus, stimulation through TCR may partially account for RANTES' unique pattern of signaling in T cells.     

Indocyanine-green videoangiography of drusen as a possible predictive indicator of exudative maculopathy

OBJECTIVE: Recent studies have shown that indocyanine-green videoangiography (ICG-V) is useful to image occult choroidal neovascularization. The authors studied the ICG-V findings in fellow drusen eyes of patients with unilateral exudative age-related macular degeneration (AMD). The authors also studied the occurrence of exudative changes to determine whether ICG-V is useful in predicting future exudative changes in these eyes with only drusen. DESIGN: Cohort study. PARTICIPANTS: The authors studied 432 consecutive patients diagnosed with unilateral exudative AMD in whom the fellow eye had only drusen by clinical fundus examination and fluorescein angiography. All of these eyes had ICG-V performed. Follow-up data were obtained in all eyes with abnormal indocyanine-green (ICG) angiograms and randomly sampled ICG angiograms of normal eyes. MAIN OUTCOME MEASURES: The initial ICG findings were classified as showing normal or abnormal hyperfluorescence. Abnormal hyperfluorescence eyes were subdivided into focal spots (focal areas of hyperfluorescence < 1 disc area in size) and plaques (areas of hyperfluorescence > 1 disc area). The development of exudative changes in eyes with normal and abnormal hyperfluorescence was compared. RESULTS: Of the 432 fellow eyes, 386 (89%) eyes with drusen had a normal ICG-V study, whereas 46 (10 focal spots and 36 plaques) (11%) eyes had an abnormal ICG-V. Exudative changes occurred in 6 (10%) of 58 normal ICG eyes and 9 (24%) of 38 eyes with abnormal ICG findings during a mean follow-up period of 21.7 months. The difference between drusen eyes with normal ICG angiograms and those with plaques on ICG-V regarding future exudative changes (10% vs. 27%, respectively) was statistically significant (P = 0.038). CONCLUSIONS: Abnormal ICG findings were found in 11% of eyes with clinically and fluorescein angiographically nonsuspicious drusen. The subgroup of patients with plaques on ICG-V had a higher chance of having exudative changes develop. Indocyanine-green videoangiography may be a predictive indicator of future exudative changes in eyes with drusen. A much larger prospective study seems justified.     

Correlates of fatigue in older women with heart failure

OBJECTIVE: To examine the relative contribution of psychologic factors and physical symptoms to the variance in fatigue in older women with heart failure. METHODS: Eighty women who had been hospitalized in the previous 12 months for heart failure were interviewed. Fifty-seven women completed second interviews 18 months after the first interview. RESULTS: Fatigue was the most frequently occurring physical symptom at both measurement times, and it significantly increased with time. Other physical symptoms contributed uniquely to the variance in fatigue at both measurement times, but psychologic factors did not. At time 1, sleep difficulties, chest pain, and weakness each explained unique variance in fatigue. At time 2, dyspnea was the only variable that explained unique variance in fatigue (9%). Dyspnea also explained a significant portion of the variance (7%) in time 2 fatigue, when time 1 fatigue was controlled. CONCLUSIONS: Fatigue in older women with heart failure is related more to other physical symptoms than psychologic factors.     

Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.     

New nonsteroidal androgen receptor modulators based on 4-(trifluoromethyl)-2(1H)-pyrrolidino[3,2-g] quinolinone

Typhoid fever is often associated with abnormal liver biochemical tests, but severe hepatic involvement with a clinical feature of acute hepatitis is a rare complication. There have been more than 150 cases of salmonella hepatitis reported from both developed and developing countries. The documented incidence varies widely from less than 1% to 26% patients with enteric fever. The possible associated factors for development of salmonella hepatitis are virulence of the organisms, delayed treatment and poor general health of the patients. The pathogenesis of severe hepatic involvement in salmonella infection may be multifactorial and includes endotoxin, local inflammatory and/or host immune reactions. Clinical jaundice in salmonella hepatitis usually occurs within the first 2 weeks of the febrile illness. Hepatomegaly and moderate elevation of transaminase levels are common findings. Extreme hepatic dysfunction with hepatic encephalopathy is a rare coexisting complication in salmonella hepatitis. A positive culture for salmonella from blood or stool is essential to differentiate salmonella hepatitis from other causes of acute hepatitis. Hepatic pathology is characterized by the presence of typhoid nodules with marked hyperplasia of reticuloendothelial cells. The prognosis is usually good as salmonella hepatitis responds well to a specific antibiotic therapy and juandice resolves with clinical improvement. The clinical course can be severe with a mortality rate as high as 20%, particularly with delayed treatment or in patients with other complications of salmonella infection. As enteric fever is a common infection, the recognition of salmonella hepatitis is of clinical importance.