Inhibition of insulin-like growth factor-I responses in MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds.

Insulin-like growth factor-I (IGF-I) stimulated the growth and [3H]thymidine uptake in MCF-7 human breast cancer cells grown in serum- and growth factor-inactivated serum-containing media. Cotreatment of the cells with IGF-I plus 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a significant decrease in mitogen-induced cell proliferation and [3H]thymidine uptake. Similar effects were observed for cells treated with 2,3,7,8-TCDD and IGF-I plus 17 beta-estradiol. The relative antimitogenic activities of 2,3,7,8-TCDD and related compounds followed the order 2,3,7,8-TCDD greater than 2,3,7,8-tetrachlorodibenzofuran (TCDF) greater than 1,2,7,8-TCDF greater than 1,3,7,8-TCDD which was similar to their aryl hydrocarbon (Ah) receptor binding affinities. The results showed that 2,3,7,8-TCDD did not alter the IGF-I receptor mRNA levels or the KD values for binding of [125I]IGF-I to the IGF-I receptor in MCF-7 cells. However, 2,3,7,8-TCDD significantly decreased the number of IGF-I-induced IGF-I receptor binding sites and this may play a role in the growth-inhibitory properties of 2,3,7,8-TCDD and related compounds and in the 'cross-talk' between the two endocrine-response pathways.     

Amphiphilic polyanhydrides for protein stabilization and release

The overall goal of this research is to design novel amphiphilic biodegradable systems based on polyanhydrides for the stabilization and sustained release of peptides and proteins. Accordingly, copolymers of the anhydrides, 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), which are monomer-containing oligomeric ethylene glycol moieties, have been synthesized. Microspheres of different CPTEG:CPH compositions have been fabricated by two non-aqueous methods: solid/oil/oil double emulsion and cryogenic atomization. The ability of this amphiphilic polymeric system to stabilize model proteins (i.e., lysozyme and ovalbumin) was investigated. The structure of both the encapsulated as well as the released protein was monitored using gel electrophoresis, circular dichroism, and fluorescence spectroscopy. It was found that the CPTEG:CPH system preserves the structural hierarchy of the encapsulated proteins. Activity studies of the released protein indicate the CPTEG:CPH system retains the biological activity of the released protein. These results are promising for future in vivo studies, which involve the design of novel biodegradable polyanhydride carriers for the stabilization and sustained release of therapeutic peptides and proteins.     

Transplant with a twist: A pitfall in sonographic diagnosis of renal transplant torsion

Torsion is an uncommon cause of impaired function in a renal transplant. We present a case of intraperitoneal transplant torsion secondary to adhesions to the left fallopian tube and ovary. Inability to confirm renal venous flow with Doppler misled to the erroneous sonographic diagnosis of renal vein thrombosis, although end diastolic flow was absent rather than reversed. The correct diagnosis was made with CT. The combination of abnormal orientation of the graft on ultrasonography, acutely impaired renal function, and abnormal Doppler study should have led to a diagnosis of transplant torsion. The case is also unusual in that the lead point was adnexal pathology. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 45 :528–530, 2017     

Effect of olsalazine on sodium-dependent bile acid transport in rat ileum

Olsalazine (OLZ), a relatively new form of 5-aminosalicylic acid (5-ASA), is being used for the treatment of colitis. A major side effect of olsalazine is diarrhea, reported in 12–25% of patients. One suggested mechanism for this side effect is enhanced ileal water and electolyte secretion. We propose that OLZ may also inhibit ileal bile acid (BA) transport, resulting in choleretic diarrhea. This would result in excess BAs reaching the colon, with consequent BA-induced secretory diarrhea. Therefore, we studied the effect of OLZ on rat ileal absorption of taurocholate. BA uptake was determined in rat ileal segments, everted sacs, brush border membrane vesicles (BBMV), andXenopus laevis oocytes. Segments and everted sacs were treated with 5 mM OLZ for 30 min prior to and throughout 10-min taurocholate (Tc) uptake. Terminal ileal BBMV were used to study the effect of OLZ on sodium-dependent bile acid uptake independent of cellular metabolism. Direct effects on the bile acid carrier were examined usingXenopus laevis oocytes expressing the cloned apical rat ileal BA transporter. In ileal segments 5 mM OLZ inhibited 10-min Tc uptake by 69.4±8.8% (P<0.01) (N=10 animals). Increasing concentrations of OLZ resulted in a dose-dependent inhibition of Tc uptake. Ten-minute Tc uptake with 0.5, 1.0, 2.0, 2.5, and 5 mM OLZ was inhibited by 13.5, 39.6, 49.7, and 70.5%, respectively. In BBMV, OLZ inhibited 45-sec Tc uptake in a dose-dependent manner but did not effect Na-dependentl-alanine uptake. Kinetic analysis revealed a noncompetitive inhibition by 2 mM olsalazine. Olsalazine, 5 mM, also inhibited Na-dependent uptake of Tc into oocytes, which expressed the rat ileal sodium-dependent bile acid transporter (8.0±3.7 vs 2.6±2.0 pmol/oocyte/hr,P<0.001). OLZ inhibits sodium-dependent Tc uptake and transmucosal transport in the rat ileum in a dose-dependent manner. This inhibition is relatively specific, noncompetitive, and does not require intact cellular mechanisms. This effect of OLZ on ileal function may contribute to the diarrhea frequently observed with this drug.Supported in part by a grant from Reach Out For Youth with IBD and a gift from Ruth & Leonard Litwin and by grants from the National Institute of Health (DK02076,34989,43509, HD07388, HD20632 and HD27757).This work was presented in part at the American Gastroenterological Association meeting, Boston, Massachusetts, May 19, 1993.Address for reprint requests: Dr. Anupama Chawla, Pediatric Gastroenterology, North Shore University Hospital-Cornell University Medical College, 300 Community Drive, Manhasset, New York 11030.     

Control of bacterial exoelectrogenesis by c-AMP-GMP

Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.Three major types of cyclic dinucleotide second messengers have been discovered in biological systems. The first to be found was c-di-GMP (Fig. 1A, Top), which serves as a regulator of diverse physiological changes in most bacterial lineages (1). The second, c-di-AMP (2), is an important signaling compound during bacterial sporulation and germination (3) and has also been found to regulate osmotic shock responses in Gram-positive bacteria (4, 5). More recently, a third type of cyclic dinucleotide called c-AMP-GMP (Fig. 1A, Bottom) was found to be required for Vibrio cholera virulence (6).Open in a separate windowFig. 1.Selective recognition of c-AMP-GMP by a natural aptamer. (A) Chemical structures of c-di-GMP and c-AMP-GMP. (B) Sequence and secondary structure of the 100 erfK RNA from G. metallireducens. P1, P2, and P3 identify base-paired substructures. M1 designates a mutant construct wherein position 20 is changed to G. Regions of constant, decreasing, and increasing RNA cleavage upon addition of ligand are indicated by yellow, red, and green circles, respectively. The arrowhead indicates the start of the RNA structure stability data derived from C. (C) PAGE analysis of in-line probing assays of 5′ ³²P-labeled 100 erfK in the presence (10 µM) of various cyclic dinucleotides. NR, T1, and ^‒OH designate lanes loaded with precursor RNA (Pre), RNA partially digested with RNase T1 (resulting in cleavage after G residues), and RNA partially digested with alkali (resulting in cleavage after every residue). Several RNase T1 cleavage product bands are labeled. Regions undergoing substantial change in spontaneous cleavage rates are labeled 1–4. The inosine-based analog of c-di-GMP is labeled c-di–IMP. (D) Plot of the fraction of riboswitch bound to ligand versus the log of the molar concentration (c) of the ligand. Data are derived from Fig. S2, and each point is the average of the normalized fraction of modulation at sites 1 and 2. Error bars indicate the SD of the average. Included are theoretical curves expected for one-to-one interaction between ligand and RNA for the K_D values given.Although bacterial c-AMP-GMP is formed via two 3′,5′-phosphodiester linkages, metazoans produce an analog with one 2′,5′-phosphodiester linkage and one 3′,5′-phosphodiester linkage (7–9). This natural structural variant has been identified as important for triggering innate immune responses in metazoans (7–11). Far greater diversity of cyclic dinucleotides is possible by varying either the nucleotide composition or the manner in which dinucleotides are joined. Therefore, many additional types of cyclic dinucleotides might await discovery in nature where they might regulate other critical cellular processes.Our approach to uncovering the chemical structures and biological functions of cyclic dinucleotide signaling compounds is to discover their RNA receptors. Three different riboswitch receptor classes have already been discovered that respond to c-di-GMP (12, 13) and c-di-AMP (5). Riboswitches are structured noncoding RNA (ncRNA) domains that selectively bind a small molecule or ion and thereby trigger a change in the expression of associated genes (14, 15). The majority of riboswitch ligands are composed of or derived from nucleotides, which supports the hypothesis that RNA-based metabolites and their riboswitch partners might descend from primordial RNA world organisms (16). If true, then cyclic dinucleotides and their receptors might be modern reflections of ancient signaling pathways. Regardless of their origins, the discovery of each new regulatory RNA motif that selectively recognizes a second messenger also reveals a superregulon for the signaling compound and the biology that it controls (5, 12, 13).Given that the diversity of natural cyclic dinucleotides might be greater than is currently known, and given that some riboswitch classes have undergone subtle changes to adapt to different ligands (17–20), we re-examined known cyclic dinucleotide riboswitch classes and their associated genes for evidence of unidentified ligands or riboswitch classes. Of special interest were those RNAs that both carry mutations in the core of the ligand-binding aptamer and have gene associations considered unusual for the parent riboswitch class. Through these efforts, we identified a subset of riboswitches within the c-di-GMP-I riboswitch class (12) that we hypothesized might bind a different ligand. In the current study, we demonstrate that these riboswitches recognize c-AMP-GMP and control a set of genes that are important for the utilization of iron(III) oxide in exoelectrogenesis.     

Intensive care in patients with HIV infection in the era of highly active antiretroviral therapy

STUDY OBJECTIVES: The use of highly active antiretroviral therapy (HAART) has dramatically improved morbidity and mortality in patients with HIV infection. The types of critical illness and their outcomes in HIV-infected patients in recent years is unknown. DESIGN: We reviewed the medical records of all patients admitted to the Medical ICU of Beth Israel Medical Center, NY, from January to June 2001 and compared their characteristics with patients admitted to the same unit from November 1991 to October 1992. RESULTS: Of 441 admissions in the first half of 2001, 63 admissions (14%) were in 53 HIV-seropositive patients. There were 65 admissions to the Medical ICU during the 1-year period spanning 1991 to 1992. Compared with the earlier period, the 2001 patients were more likely to be black (52% vs 26%, respectively; p < 0.01) and injection drug users (75% vs 48%, respectively; p < 0.01), and were less likely to be white (11% vs 23%, respectively; difference not significant) and homosexual men (6% vs 26%, respectively; p < 0.01). In 2001, patients were less likely to be admitted with respiratory failure (22% vs 54%, respectively; p < 0.01) and with Pneumocystis jiroveci pneumonia (formerly referred to as Pneumocystis carinii) [3% vs 34%, respectively; p < 0.001], and were more likely to be admitted with non-HIV-related diseases (67% vs 12%, respectively; p < 0.001). Overall survival was much higher in the later period (71% vs 49%, respectively; p < 0.01). CONCLUSIONS: In the era of HAART, more patients with HIV infection were admitted to the ICU over a 12-month period than were 10 years previously. Patients were more likely to be injection drug users and were more likely to be admitted to the ICU because of non-HIV-associated conditions.     

Effects of amiodarone on supraventricular tachycardia involving bypass tracts

This study evaluates whether the electrophysiologic effects of i.v. amiodarone in patients with reentrant supraventricular tachycardia (SVT) can predict the efficacy of long-term oral therapy with this drug. The effects of oral and i.v. amiodarone were studied in 27 patients with SVT. In 14 the SVT circuit involved a concealed atrioventricular bypass for retrograde conduction (Group I), and in 13 a concealed atrio-His bypass (Group II). Intravenous amiodarone induced significant prolongation of the AH interval, the refractory periods of the atrium, atrioventricular node, His-Purkinje system and ventricular myocardium. The ventriculoatrial interval was slightly prolonged in Group I patients and did not change in Group II patients after i.v. administration of the drug. In both groups, the effective refractory period (ERP) of the concealed bypass was prolonged by i.v. amiodarone.During control state, SVT could be induced in all patients; after i.v. administration of the drug, SVT was presented in 6 patients in Group I and in 8 patients in Group II. In all cases, in which i.v. amiodarone prolonged the ERP of the concealed bypass to more than 350 ms, the drug always prevented SVT even when given orally. All but 2 patients—1 from Group I and 1 from Group II—remained asymptomatic after oral amiodarone. In the patient from Group I, SVT had been prevented by i.v. amiodarone, whereas in the patient from Group II SVT could not be induced by ventricular stimulation during the control state, but appeared after i.v. administration of the drug.These data indicate that (1) therapy with oral amiodarone is effective in most patients with SVT irrespective of the type of bypass fibers, i.e., atrio-His or Kent bundle; (2) effective suppression of induction of SVT with i.v. amiodarone is a predictor of efficacy of amiodarone in 93 % of the cases. However, a lack of response to i.v. amiodarone is not a predictor of lack of efficacy of oral amiodarone; (3) prolongation of the ERP of the concealed bypass tract to > 350 ms appears to have a favorable predictive value in oral administration; (4) a change in the inducibility of SVT after i.v. amiodarone appears to have a negative predictive value in controlling SVT with the oral drug.