A single phenylalanine residue in β-arrestin2 critically regulates its binding to G protein–coupled receptors

Arrestins and their yeast homologs, arrestin-related trafficking adaptors (ARTs), share a stretch of 29 amino acids called the ART motif. However, the functionality of that motif is unknown. We now report that deleting this motif prevents agonist-induced ubiquitination of β-arrestin2 (β-arr2) and blocks its association with activated G protein–coupled receptors (GPCRs). Within the ART motif, we have identified a conserved phenylalanine residue, Phe116, that is critical for the formation of β-arr2–GPCR complexes. β-arr2 Phe116Ala mutant has negligible effect on blunting β₂-adrenergic receptor–induced cAMP generation unlike β-arr2, which promotes rapid desensitization. Furthermore, available structures for inactive and inositol hexakisphosphate 6–activated forms of bovine β-arr2 revealed that Phe116 is ensconced in a hydrophobic pocket, whereas the adjacent Phe117 and Phe118 residues are not. Mutagenesis of Phe117 and Phe118, but not Phe116, preserves GPCR interaction of β-arr2. Surprisingly, Phe116 is dispensable for the association of β-arr2 with its non-GPCR partners. β-arr2 Phe116Ala mutant presents a significantly reduced protein half-life compared with β-arr2 and undergoes constitutive Lys-48-linked polyubiquitination, which tags proteins for proteasomal degradation. We also found that Phe116 is critical for agonist-dependent β-arr2 ubiquitination with Lys-63-polyubiquitin linkages that are known mediators of protein scaffolding and signal transduction. Finally, we have shown that β-arr2 Phe116Ala interaction with activated β₂-adrenergic receptor can be rescued with an in-frame fusion of ubiquitin. Taken together, we conclude that Phe116 preserves structural stability of β-arr2, regulates the formation of β-arr2–GPCR complexes that inhibit G protein signaling, and promotes subsequent ubiquitin-dependent β-arr2 localization and trafficking.     

Acute and subchronic toxicity of aflatoxin B1 to rohu, Labeo rohita (Hamilton)

Pathological alterations in various organs of rohu (L. rohita) fingerlings following acute (0, 7.50, 11.25 and 13.75 mg/kg body weight) and subchronic (0, 1.25 and 2.50 mg/kg body weight) single i.p. aflatoxin B1 exposure for 10 and 90 days, respectively, were investigated. Mortality (dose-dependent) was marked only during acute toxicosis. The changes observed in various organs were dose and time dependent. The acute dose groups revealed toxic changes viz., necrotic and vascular changes in liver and gill lamellae; meningitis, congestion in brain, degeneration and inflammatory reaction in heart along with degenerative to necrotic changes in kidney tubules and sloughing of the intestinal mucosa. During subchronic exposure to this toxin, preneoplastic lesions in liver along with changes in spleen, intestine, gill and pancreas were recorded. With low doses of aflatoxin, the fish did not reveal any mortality or external signs other than catchexia and increased pigmentation on scales. In composite culture practice of Indian major carps, this could be of economic significance.     

Leishmanicidal activity of stearylamine-bearing liposomes in vitro

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.     

Exploring the interaction of some N-benzyloxycarbonyl-L-phenyl alanyl-L-alanine ketones and bovine spleen cathepsin B by molecular modeling and binding free energy calculation

A semi-empirical method for estimation of binding free energy, recently proposed by Aqvist and coworkers, has been effectively tested in several protein-ligand binding cases. We have applied this linear interaction energy method to predict the binding of some N-benzyloxycarbonyl-L-phenyl alanyl-L-alanine ketones with bovine cathepsin B and computed the respective absolute binding constants from averages of molecular dynamics simulations. It is found that the computer simulation results agree well with available experimental data and make it possible to understand better the origin of tight binding and inhibitor specificity of cathepsin B.     

Fractal dimension in aspiration cytology smears of breast and cervical lesions

OBJECTIVE: To standardize the automated measurement of fractal dimension on cytologic smears and compare the fractal dimension of benign and malignant breast cells and cervical lesions on cytologic material to evaluate its role in the discrimination of benign from malignant cells. STUDY DESIGN: We randomly selected fine needle aspiration cytology smears of 42 cases of infiltrating duct carcinoma and 38 cases of fibroadenoma of the breast. Similarly, 16 cervical carcinoma and 20 normal cervical smears were selected for study. Ten cells were selected randomly from each case. Box counting of fractal dimension of malignant and benign cells was achieved with an image cytometer (Leica, Cambridge, England) using Quantimet 600 software (Leica). Then a well-spaced grid with multiple small boxes of a particular pixel length was superimposed on the cell. The dimension of the box was selected as 4, 8 and 16 pixels. With the help of a logical "AND" operation, we counted the number of boxes touching the peripheral margin of the cell nuclei. For each cell, the log-log graph of 1 per box size was plotted against the number of boxes touching the peripheral rim of the cell. The slope of each graph was identified using the least-squares method of regression analysis. RESULTS: The mean fractal dimension of malignant cells was 0.8536 +/- 0.1120 as compared to 0.8403 +/- 0.1115 in benign cell groups. The Mann-Whitney U test showed a significant difference in fractal dimension in these 2 groups (P = .05). The mean fractal dimension of malignant cells from the cervix was 0.8656 +/- 0.1499 as compared to 0.8315 +/- 0.1312 in benign cells. The Mann-Whitney U test showed a significant difference in fractal dimension in these 2 groups (P < .02). CONCLUSION: Fractal dimension may be a helpful adjunctive technique to discriminate between benign and malignant cells.     

Fractal dimension in endometrial carcinoma

OBJECTIVE: To mathematically assess in a pilot study, endometrial glandular margin irregularity in simple hyperplasia, complex atypical hyperplasia and well-differentiated endometrial carcinoma with the help of box counting of fractal dimension and to discriminate these lesions on the basis of box counting of fractal dimension of the gland. STUDY DESIGN: Ten cases each of endometrial simple hyperplasia (without atypia), complex hyperplasia with atypia and endometrial carcinoma (well-differentiated, endometrioid) were assessed in the study. Five fields at 20 x magnification from each case were randomly selected, and the glands were outlined with the help of a pointer. Using the box counting method, the fractal dimension of each case was measured. RESULTS: Mean fractal dimension in simple hyperplasia, complex atypical hyperplasia and endometrial carcinoma was, 0.899 +/- 0.13, 0.932 +/- 0.042 and 0.939 +/- 0.02, respectively. Statistical analysis showed that the fractal dimension of glands of simple hyperplasia were significantly different from that of complex atypical hyperplasia and endometrial carcinoma (P = .041 and .013, respectively, ANOVA). However, there was no significant difference in fractal dimension between glands of complex hyperplasia and of endometrial carcinoma (P = .659, ANOVA). CONCLUSION: This study provides mathematical (objective) assessment of the measurement of glandular margin irregularities in simple hyperplasia, complex atypical hyperplasia and endometrial carcinoma. Fractal dimension of gland margin may have diagnostic potential in the future.     

Uterine Msx-1 and Wnt4 signaling becomes aberrant in mice with the loss of leukemia inhibitory factor or Hoxa-10: evidence for a novel cytokine-homeobox-Wnt signaling in implantation

Successful implantation absolutely depends on the reciprocal interaction between the implantation-competent blastocyst and the receptive uterus. Expression and gene targeting studies have shown that leukemia inhibitory factor (LIF), a cytokine of the IL-6 family, and Hoxa-10, an abdominalB-like homeobox gene, are crucial to implantation and decidualization in mice. Using these mutant mice, we sought to determine the importance of Msx-1 (another homeobox gene formerly known as Hox-7.1) and of Wnt4 (a ligand of the Wnt family) signaling in implantation because of their reported functions during development. We observed that Msx-1, Wnt4, and a Wnt antagonist sFRP4 are differentially expressed in the mouse uterus during the periimplantation period, suggesting their role in implantation. In addition, we observed an aberrant uterine expression of Msx-1 and sFRP4 in Lif mutant mice, and of Wnt4 and sFRP4 in Hoxa-10 mutant mice, further reinforcing the importance of these signaling pathways in implantation. Collectively, the present results provide evidence for a novel cytokine-homeotic-Wnt signaling network in implantation.     

Multiple connections link FAK to cell motility and invasion

The ability of intracellular signaling networks to orchestrate a complex biological response such as cell motility requires that individual signaling proteins must act as integrators, responding to multiple extracellular inputs and regulating multiple signaling pathway outputs. In this review, we highlight recent findings that place focal adhesion kinase (FAK) in an important receptor-proximal position in the regulation of growth factor and integrin-stimulated cell motility. Emphasis is placed on the molecular mechanisms of FAK activation, connections of FAK to focal contact formation as well as turnover, and the potential that FAK function in promoting cell invasion may be distinct from its role in cell motility.     

Nanoscale organization of multiple GPI-anchored proteins in living cell membranes

Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosyl-phosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments, combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.