On the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides

BACKGROUND: Cell-permeable small molecules that target predetermined DNA sequences with high affinity and specificity have the potential to control gene expression. A binary code has been developed to correlate DNA sequence with side-by-side pairings between N-methylpyrrole (Py) and N-methylimidazole (Im) carboxamides in the DNA minor groove. We set out to determine the relative energetics of pairings of Im/Py, Py/Im, Im/Im, and Py/Py for targeting G.C and A.T base pairs. A key specificity issue, which has not been previously addressed, is whether an Im/Im pair is energetically equivalent to an Im/Py pair for targeting G.C base pairs. RESULTS: Equilibrium association constants were determined at two five-base-pair sites for a series of four six-ring hairpin polyamides, in order to test the relative energetics of the four aromatic amino-acid pairings opposite G.C and A.T base pairs in the central position. We observed that a G.C base pair was effectively targeted with Im/Py but not Py/Im, Py/Py, or Im/Im. The A.T base pair was effectively targeted with Py/Py but not Im/Py, Py/Im, or Im/Im. CONCLUSIONS: An Im/Im pairing is energetically disfavored for the recognition of both A.T and G.C. This specificity will create important limitations on undesirable slipped motifs that are available for unlinked dimers in the minor groove. Baseline energetic parameters will thus be created which, using the predictability of the current pairing rules for specific molecular recognition of double-helical DNA, will guide further second-generation polyamide design for DNA recognition.     

Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs)

IAPs comprise a family of inhibitors of apoptosis found in viruses and animals. In vivo binding studies demonstrated that both baculovirus and Drosophila IAPs physically interact with an apoptosis-inducing protein of Drosophila, Reaper (RPR), through their baculovirus IAP repeat (BIR) region. Expression of IAPs blocked RPR-induced apoptosis and resulted in the accumulation of RPR in punctate perinuclear locations which coincided with IAP localization. When expressed alone, RPR rapidly disappeared from the cells undergoing RPR-induced apoptosis. Expression of P35, a caspase inhibitor, also blocked RPR-induced apoptosis and delayed RPR decline, but RPR remained cytoplasmic in its location. Mutational analysis of RPR demonstrated that caspases were not directly responsible for RPR disappearance. The physical interaction of IAPs with RPR provides a molecular mechanism for IAP inhibition of RPR's apoptotic activity.     

Occupational allergic contact dermatitis in a patient with a positive patch test to tin

The case of a 69-year-old man with liver cirrhosis, thrombocytopenia, unstable angina, and a history of previous coronary artery bypass grafting (CABG) is presented. The patient under-went successful repeat CABG through lateral thoracotomy on the beating heart without extracorporeal circulatory support.     

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3((1-87))), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.     

Acid secretion from an esophageal inlet patch demonstrated by ambulatory pH monitoring

Heterotopic gastric mucosa may occur throughout the gastrointestinal tract, including the upper esophagus. The capability of this ectopic mucosa to secrete acid has been suggested in different reports. We report for the first time a case of heterotopic gastric mucosa in the upper esophagus complicated by a stricture with secretion of acid demonstrated by prolonged ambulatory pH monitoring. Lansoprazole, 30 mg twice daily, produced symptom resolution, and repeat ambulatory pH showed complete acid suppression in the proximal esophagus.     

Inhibition of Salmonella typhimurium invasion by host cell expression of secreted bacterial invasion proteins

Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the role of secreted proteins as direct modulators of invasion, we have evaluated the ability of Salmonella typhimurium to enter mammalian cells that express portions of the Salmonella invasion proteins SipB and SipC. Plasma membrane localization of SipB and SipC was achieved by fusing carboxyl- and amino-terminal portions of each invasion protein to the intracellular carboxyl-terminal tail of a membrane-bound eukaryotic receptor. Expression of receptor chimeras possessing the carboxyl terminus of SipB or the amino terminus of SipC blocked Salmonella invasion, whereas expression of their chimeric counterparts had no effect on invasion. The effect on invasion was specific for Salmonella since the perturbation of uptake was not extended to other invasive bacterial species. These results suggest that Salmonella invasion can be competitively inhibited by preventing the intracellular effects of SipB or SipC. In addition, these experiments provide a model for examining interactions between bacterial invasion proteins and their host cell targets.     

The theoretical limits of DNA sequence discrimination by linked polyamides

Linked polyamides bind in the minor groove of double-stranded DNA in a partially sequence-specific manner. This report analyzes the theoretical limits of DNA sequence discrimination by linked polyamides composed of two to four different types of heterocyclic rings, determining (i) the optimal choice of base-binding specificity for each ring and (ii) the optimal design for a polyamide composed of these rings to target a given DNA sequence and designed to maximize the fraction of the total polyamide binding to the specified target sequence relative to all other sequences. The results show that, fortuitously, polyamides composed of pyrrole, a naturally occurring G-excluding element, and imidazole, a rationally designed G-favoring element, have features similar to the theoretical optimum design for polyamides composed of two different rings. The results also show that, in polyamides composed of two or three types of heterocyclic rings, choosing a nonspecific "placeholder" ring, which binds equally strongly to each of the four bases, along with one or two base-specific rings will often enhance sequence specificity over a polyamide composed entirely of base-specific rings.     

Inhibition of intestinal motility by anandamide, an endogenous cannabinoid

The endogenous cannabinoid ligand anandamide (arachidonylethanolamide) inhibited the intestinal passage of a charcoal meal when administered s.c. in mice at doses ranging from 0.1 to 50 mg/kg. This effect was prevented by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide x HCl] (1 mg/kg s.c.), but it was not affected by the anandamide transport inhibitor, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (50 mg/kg, s.c.). The results indicate that anandamide modulates intestinal motility in mice by activating cannabinoid CB1 receptors. They also suggest that anandamide transport, which was previously shown to participate in terminating neural and vascular responses to anandamide, does not contribute to anandamide inactivation in intestinal tissue.     

Stricture related to an inlet patch of the esophagus

A 64-yr-old female was referred for evaluation of dysphagia. An esophagram showed a stricture in the proximal esophagus. On esophagogastroduodenoscopy, the patient was found to have a 3-cm stricture beginning at 14 cm from the incisors. This was initially dilated to 45-French with the Savary-Gilliard dilators. Biopsies and cytology of the stricture showed benign columnar epithelium. Subsequent dilations over the next 2 months resulted in dilation to 60-French with the esophageal dilators. Ulcerations related to this strictured area were healed over this period, with a sucralfate slurry, and the area of abnormality after healing appeared to be that of an esophageal inlet patch.     

Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins

Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.     

Inhibition of nucleolar protein nucleolin by electroporation with anti-nucleolin antibodies results in an increase of the nucleolar size

We produced an antibody that recognized only early stages of spermatogonia in Japanese eel testis. This antibody (anti-spermatogonia-specific antigen-1, anti-SGSA-1) recognized a band of about 38 kDa in Western blot analysis of extracts from eel testis. This antigen was observed by immunohistochemistry only in type-A and early type-B spermatogonia and could not be seen in the late type-B spermatogonia, which appeared after the initiation of spermatogenesis by a single injection of human chorionic gonadotropin. Immunoreactive SGSA-1 was absent in spermatocytes, spermatids, spermatozoa, Sertoli cells, and interstitial Leydig cells. Similarly, this antigen was also detected only in type-A/primary spermatogonia in the testes of two species of teleosts, medaka (Oryzias latipes) and tilapia (Oreochromis niloticus), as well as a toad (Xenopus laevis). These results imply that the disappearance of SGSA-1 in late type-B/secondary spermatogonia is a critical step in the progression of spermatogenesis, and indicate that anti-SGSA-1 is a useful marker for analysis of the molecular mechanism controlling the differentiation of spermatogonia in lower vertebrates.     

Histidine patch thioredoxins. Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins

A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for convenient purifications of human interleukin-8 and -11 expressed in E. coli as soluble thioredoxin fusion proteins.     

Inhibition of gastric acid secretion by blood drawing from an indwelling venous needle

To investigate a possible inhibitory effect of blood drawing through an indwelling forearm vein needle on gastric acid secretion, 11 subjects were studied on four occasions each. The first session was for adapting the subject to the 3-hr collection of gastric juice. In 7 subjects the second through fourth sessions gave three conditions in balanced order: (1) an indwelling forearm vein needle and the withdrawal of 5 or 10 cc of blood every 20 min, (2) only a nonfunctional "dummy" needle implanted subcutaneously in the forearm skin, and (3) the control condition with no needle. In four additional subjects the sessions were identical except that condition (1) involved an indwelling forearm vein needle kept open by a slow infusion of saline solution and no blood was drawn. Phenol red recovery from an initial intragastric injection was measured in all. Results showed that blood drawing, but not saline infusion or venipuncture per se, inhibited gastric acid output.     

A Study of Aluminium Corrosion Inhibition in Acid Medium by an Antiemitic Drug

The inhibition effect of meclizine hydrochloride on the corrosion behaviour of aluminium in 1 M hydrochloric acid medium was studied by weight loss, potentiodynamic polarisation and electrochemical impedance spectroscopy techniques. Results obtained revealed that Meclizine performed excellently as a corrosion inhibitor for aluminium in HCl medium at 303 K. The effect of temperature on inhibition efficiency was studied. The inhibition was assumed to occur via adsorption of the inhibitor molecule on the metal surface. The adsorption of the inhibitor on the aluminium surface followed Langmuir adsorption isotherm model. The values of activation energy and thermodynamic parameters were calculated and discussed.     

Inhibition of Escherichia coli heat-labile enterotoxin by an amino carbonyl product, lactose-alpha-lactalbumin

The inhibition by lactose-alpha-lactalbumin amino carbonyl product of Escherichia coli heat-labile enterotoxin was studied by GM1-ELISA and by assay with CHO-K1 cells. The product dose-dependently inhibited the binding of the enterotoxin to GM1 ganglioside and decreased the morphological change of CHO-K1 cells caused by this toxin. The results suggest that this product may be a receptor analogue in the intestine.