Utility and impact of early t(15;17) identification by Fluorescence In Situ Hybridization (FISH) in clinical decision making for patients in Acute Promyelocytic Leukemia (APL).

INTRODUCTION: Acute Promyelocytic Leukemia (APL) is a curable malignancy with studies showing above 90% survival. However, population-based studies looking at survival suggest that approximately 30% of patients with APL die during induction. Early demonstration of t(15;17) will lead to accurate decision making regarding treatment. The aim of this project was to validate earlier time frames for the Abbott Molecular Vysis LSI promyelocytic leukemia (PML)/ retinoic acid receptor alpha (RARA) fluorescence in situ hybridization (FISH) probe (ASR 6-16 h). METHODS: Twenty patients (15 APL cases and five non-APL cases) were selected for validating various hybridization times for the FISH probe. Expected normal signal pattern was two red and two green signals (2R2G), and the most common expected abnormal signal pattern was two fusion (yellow) signals, one red and one green (2F1R1G) and/or one fusion, one red and one green (1F1R1G). RESULTS: The specificity of the probe ranged from 84% at 2 h, 86% at 4 h, 84% at 6 h, and 87% for overnight hybridization. The sensitivity increased from 79% at 2 h, 80% at 4 h, 81% at 6 h to 87% for overnight hybridization. CONCLUSION: Based on the validation studies, we recommend reading of FISH results at the 4-h incubation mark for a preliminary diagnosis and confirmation with overnight hybridization.

Evidence for hydrophobic interaction between galanin and the GalR1 galanin receptor and GalR1-mediated ligand internalization: fluorescent probing with a fluorescein-galanin.

Galanin is a neuropeptide that activates specific receptors to modulate several physiological functions including food intake, nociception, and learning and memory. The molecular nature of the interaction between galanin and its receptors and the fate of the galanin/receptor complex after the binding event are not understood. A fluorescein-N-galanin (F-Gal) was generated to measure the interaction between galanin and rat GalR1 galanin receptor (rGalR1) and rGalR1-mediated ligand internalization using flow cytometry in transfected Chinese hamster ovary (CHO) cells. Like galanin, F-Gal bound rGalR1 with high affinity and stimulated intracellular signaling events. Fluorescence quenching by soluble KI of rGalR1-bound F-Gal revealed a highly protected environment around the fluorescein, suggesting that the N-terminal portion of galanin, which constitutes the binding site of galanin for the receptor, binds to a protected hydrophobic binding pocket within the receptor. Exposure to F-Gal stimulated rapid (t1/2 approximately 10 min) and extensive (78%) internalization of surface F-Gal into rGalR1/CHO cells at 37 degreesC but not at 0 degreesC. In addition, the internalization did not occur in parental CHO cells at either 0 or 37 degreesC and was inhibited by addition of 0.25 M sucrose in the medium, indicating a GalR1-mediated energy-requiring endocytic process. These results revealed a hydrophobic interaction between galanin and the GalR1 receptor, which is in contrast to those of other G protein-coupled receptors that mainly require hydrophilic interaction with their peptide ligands near or outside the plasma membrane surface, and illustrated that the initial binding interaction is followed by rapid cellular internalization of the agonist/GalR1 complex.

Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes.

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.

Retrovirus-mediated expression of the GalR1 galanin receptor: implication for efficient stable expression of functional G protein-coupled receptors.

The rat GalR1 galanin receptor was used as a prototypic G protein-coupled receptor to test the feasibility of heterologous expression in a retrovirus-based system. The system utilizes an independent retroviral vector pMX, a virus-packaging cell line BOSC23 and a pre-B cell line BA/F3 as the host for expression. A polyclonal cell population that expresses high ligand affinity (KD = 0.18 nM) and high level (7 pmol/mg) of GalR1 was generated within days with no drug sensitivity-based selection. The expression represented a 20-fold increase over the expression level of GalR1 achieved in CHO cells. The affinity of galanin for the expressed receptor was decreased by 19-fold in the presence of GTP-gamma-S, suggesting that the expression system can produce active galanin receptor functionally coupled to G proteins. The fast and efficient method to generate stable cell lines and to prepared large quantities of receptors may provide a general application for expression of other G protein-coupled receptors.

Wiedendiol-A inhibits cholesteryl ester binding to its transfer protein.

AIM: To study the wiedendiol-A (W-A) inhibition mechanism of plasma cholesteryl ester (CE) transfer protein (CETP) on the transfer of CE. METHODS: Using gel filtration method. RESULTS: W-A at 30 mumol.L-1 inhibited association of CE with CETP by 76% and CETP transfer activity by 81%. In addition, W-A enhanced binding of TP2, a monoclonal antibody with a CETP C-terminal epitope which is involved in CE binding, to CETP, suggesting a W-A-induced conformational change at the epitope for increased TP2 binding. When CETP activity was measured by varying high-density lipoproteins (HDL) concentration, the apparent Vmax of CE transfer was inhibited by 74% and 83% in the presence of W-A at 14 and 25 mumol.L-1, respectively, while the apparent K(m) of HDL for CETP did not change. CONCLUSION: W-A action is mediated through interaction between W-A and CETP, but not through those between W-A and lipoproteins.

Genomic organization and functional characterization of the mouse GalR1 galanin receptor.

Galanin mediates diverse physiological functions in digestive, endocrine, and central nervous systems through G-protein-coupled receptors. Two galanin receptors have been cloned but the gene structures are unknown. We report genomic and cDNA cloning of the mouse GalR1 galanin receptor and demonstrate that the coding sequence is uniquely divided into three exons encoding the N-terminal portion through the fifth transmebrane domain, the third intracellular loop, and the sixth transmembrane domain through the C-terminus. Functional analysis of the encoded cDNA revealed active ligand binding and intracellular signaling. The expression is detected in brain, spinal cord, heart and skeletal muscle.

Local initiation of spermatogenesis in the horse.

Gross observation of testicular parenchyma of 1.5- to 2-yr-old horses reveals both light and dark regions. If this gross, differential shading reflects quantitative differences in the development of spermatogenesis and interstitial cell populations, the horse may prove to be a useful model for study of the paracrine relationships associated with initiation of spermatogenesis. The objective of this study was to characterize seminiferous tubules and interstitium of testes with gross, differential shading. Testes with both light and dark regions of parenchyma were obtained from horses 1.5-2 yr old and compared to parenchyma of fetal, 2-yr-old, or 5-yr-old horses. Stereology was used on tubular and interstitial components, and luminal development of seminiferous tubules was scored. Volume density of seminiferous tubules, percentage of tubules with large vacuoles or a complete lumen, and number of primary spermatocytes per gram were greater (p < 0.05) in light parenchyma than in dark parenchyma. The percentage of tubules with no lumen and the percentage of parenchyma occupied by interstitial space were greater (p < 0.05) in fetal and dark parenchyma than in light parenchyma. The number of Leydig cells per gram parenchyma was similar (p > 0.05) in both light parenchyma and dark parenchyma. A greater percentage (p < 0.05) of other (nonvascular, non-Leydig, nonmacrophage) cells was found in the dark parenchyma than in light parenchyma or in testes of 2- or 5-yr-old horses. The volume density of macrophages was notably greater (p < 0.05) in fetal and dark parenchyma than in light parenchyma or in testes from older horses. Variation in development of seminiferous tubules was not associated with the volume density of blood vessels. In conclusion, the gross, differential shading of equine testicular parenchyma with its corresponding differences in seminiferous tubular development is a clear example of the effect of local factors leading to the local initiation of spermatogenesis.

Hypocholesterolemic activity of a novel inhibitor of cholesterol absorption, SCH 48461.

The amount of cholesterol that circulates in the plasma as lipoproteins can be affected by the balance of cholesterol metabolism within and between the intestines and liver. In the present report, we describe a novel hypocholesterolemic agent and document its pharmacological effects in animal models of hypercholesterolemia. The oral administration of (3R,4S)-1,4-bis-(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone (SCH 48461) reduced plasma cholesterol concentrations in cholesterol-fed hamsters, rats and rhesus monkeys with ED50s of 1, 2 and 0.2 mg/kg per day, respectively, SCH 48461 was also highly effective in reducing hepatic cholesteryl ester accumulation in cholesterol-fed hamsters and rats after 7 days of treatment. In one 3 week study, rhesus monkeys were fed a 0.25% cholesterol/22% saturated fat diet with or without SCH 48461. At the end of the 3 week period the control group's VLDL + LDL-cholesterol increased to 180 Mg/dl from a baseline of approximately 65 mg/dl while plasma apolipoprotein B levels had doubled. Animals treated daily with 1 mg/kg SCH 48461 maintained their baseline levels of VLDL + LDL-cholesterol, HDL-cholesterol, and plasma apolipoproteins B and A-I. After 3 weeks the diets of the two groups were switched. Within 1 week SCH 48461 (1 mg/kg per day) rapidly reversed the elevated VLDL + LDL-cholesterol levels of the previous control group to near baseline values. SCH 48461 exerted its hypocholesterolemic effect through the inhibition of cholesterol absorption. A dose of 10 mg/kg per day inhibited cholesterol absorption in cholesterol-fed hamsters by 68% while a similar reduction was achieved in chow-fed monkeys with 3 mg/kg per day. This latter dose inhibited cholesterol absorption in cholesterol-fed monkeys by 95%. Treatment of cholesterol-fed monkeys with 10 mg/kg per day SCH 48461 significantly increased fecal neutral sterol excretion (52 vs. 32 mg/kg) but had no effect on acidic sterol excretion. Using a 2-h absorption model in cholesterol-fed hamsters, SCH 48461 caused a 46% inhibition of unesterified [14C]cholesterol accumulation in the intestinal wall and a 90% inhibition of cholesteryl ester formation at a dose of 10 mg/kg. Similar data were observed when the plasma radioactivity was assessed, indicating inhibition of both free (61%) and esterified (85%) cholesterol appearance. In contrast, CI-976, a potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, did not affect the uptake of free cholesterol into the intestines while inhibiting cholesterol esterification (98% inhibition).(ABSTRACT TRUNCATED AT 400 WORDS)