Study of molecular mechanisms of pro-apoptotic activity of NCX 4040, a novel nitric oxide-releasing aspirin, in colon cancer cell lines

Background  

Despite numerous studies aimed at verifying the antitumor activity of nitric oxide-releasing nonsteroidal antiflammatory drugs (NO-NSAIDs), little is known about the molecular targets responsible for their antineoplastic properties. In the present study, we investigated the mechanisms underlying the cytotoxicity of NCX 4040, a novel NO-aspirin with promising antineoplastic action, in in vitro human colon cancer models.     

Synthesis of amphiphilic star-shaped and graft copolymers based on polystyrene and poly(ethylene oxide)

Amphiphilic block macromonomers possessing a central unsaturation were synthesized by condensation of polystyrene half-ester of maleic acid {α-[2-(3-carboxyacryloyloxy)ethyl]-ω-sec-butylpoly[1-phenylethylene]} with poly(ethylene glycol) monoether or polystyrene-block-poly(ethylene oxide). In the radical monomer cis-trans-isomerization homopolymerization of the diblock macromonomers, four-to eight-armed amphiphilic star-shaped copolymers were obtained. Radical copolymerization of the diblock macromonomers with styrene led to graft copolymers with low degree of grafting. The triblock macromonomers proved to be unable to polymerize.     

Localizing a putative mutation as the major contributor to the development of sporadic Hirschsprung disease to the RET genomic sequence between the promoter region and exon 2

Hirschsprung disease (HSCR), a congenital disorder characterized by intestinal obstruction due to absence of enteric ganglia along variable lengths of the intestinal tract, occurs both in familial and sporadic cases. RET mutations have been found in approximately 50% of the families, but explains only a minority of sporadic cases. This study aims at investigating a possible role of RET in sporadic HSCR patients. Haplotypes of 13 DNA markers, within and flanking RET, have been determined for 117 sporadic HSCR patients and their parents. Strong association was observed for six markers in the 5' region of RET. The largest distortions in allele transmission were found at the same markers. One single haplotype composed of these six markers was present in 55.6% of patients versus 16.2% of controls. Odds ratios (ORs) revealed a highly increased risk of homozygotes for this haplotype to develop HSCR (OR>20). These results allowed us to conclude that RET plays a crucial role in HSCR even when no RET mutations are found. An unknown functional disease variant(s) with a dosage-dependent effect in HSCR is likely located between the promoter region and exon 2 of RET.     

Identification and distribution ofEchinostoma lindoense,E. audyi andE. revolutum (Trematoda; Echinostomatidae)

The closely relatedEchinostoma lindoense, E. audyi andE. revolutum can be differentiated by morphological characteristics of their adults and cercariae. We have foundE. lindoense andE. audyi in Southeast and Southwest Asia and Central Europe and the former species also in South America. However, using the morphological characteristics described by Beaver (1937) forE. revolutum which is assumed to be cosmopolitan, we did not find this species in these regions.     

Short-term and long-term stimulation of Na+−H+ exchange in cortical brush-border membranes during compensatory growth of the rat kidney

The effect of unilateral nephrectomy on Na⁺–H⁺ exchange in rat renal cortical brush-border membrane vesicles (BBMV) was studied by the method of acridine orange fluorescence quenching. The exchanger activity in BBMV from remnant kidney increased rapidly by 70–75% within first 30 min following uninephrectomy. Only a slight further increase was found in later stages of renal growth, i.e. 30 min to 7 days following uninephrectomy. The changes in antiporter activity were restricted toV _max, whereas theK _m for Na⁺ was similar in control and compensatory growing kidney. The increase of Na⁺–H⁺ exchange at 15 min was not affected by actinomycin D in vivo, whereas the increase at 48 h was completely abolished indicating that protein synthesis could be involved in the late, but not in the initial stimulation of renal Na⁺–H⁺ exchange. The late, but not the initial stimulations of Na⁺–H⁺ exchange were associated with elevated activities of cortical (Na⁺+K⁺)-ATPase indicating that changes in antiporter activity precede those in the (Na⁺+K⁺)-pump. The early stimulation of Na⁺–H⁺ exchange in BBMV in one kidney was induced also by the occlusion of blood flow through the contralateral kidney for 15 min, without removing it. Thirty min after the occlusion was removed and the reflow established, the Na⁺–H⁺ exchange in BBMV from the intact kidney decreased to the control values. The observed modulations in renal Na⁺–H⁺ exchanger may be regulated by phosphorylation-dephosphorylation events. In support, the concentration of a well known protein kinase C activator, 1,2-diacylglycerol, in the cortical tissue of the remnant kidney increased up to 100% within 5 min following unilateral nephrectomy and preceded the increase in Na⁺–H⁺ exchange. The early stimulation of Na⁺–H⁺ exchange may be a trigger in initiating the kidney growth.     

Heart development in the spotted dolphin (Stenella attenuata)

Marine mammals show many deviations from typical mammalian characteristics due to their high degree of specialization to the aquatic environment. In Cetaceans, some of the features of limbs and dentition resemble very ancestral patterns. In some species, hearts with a clearly bifid apex (a feature normally present during mammalian embryogenesis prior to completion of ventricular septation) have been described. However, there is a scant amount of data regarding heart development in Cetaceans, and it is not clear whether the bifid apex is the rule or the exception. We examined samples from a unique collection of embryonic dolphin specimens macroscopically and histologically to learn more about normal cardiac development in the spotted dolphin. It was found that during the dolphin's 280 days of gestation, the heart completes septation at about 35 days. However, substantial trabecular compaction, which normally occurs in chicks, mice, and humans at around that time period, was delayed until day 60, when coronary circulation became established. At that time, the apex still appeared bifid, similarly to early fetal mouse or rat hearts. By day 80, however, the heart gained a compacted, characteristic shape, with a single apex. It thus appears that the bifid apex in the adult Cetacean heart is probably particular to certain species, and its significance remains unclear.     

Mutations of the GREAT gene cause cryptorchidism

In humans, failure of testicular descent (cryptorchidism) is one of the most frequent congenital malformations, affecting 1-3% of newborn boys. The clinical consequences of this abnormality are infertility in adulthood and a significantly increased risk of testicular malignancy. Recently, we described a mouse transgene insertional mutation, crsp, causing high intraabdominal cryptorchidism in homozygous males. A candidate gene Great (G-protein-coupled receptor affecting testis descent), was identified within the transgene integration site. Great encodes a seven-transmembrane receptor with a close similarity to the glycoprotein hormone receptors. The Great gene is highly expressed in the gubernaculum, the ligament that controls testicular movement during development, and therefore may be responsible for mediating hormonal signals that affect testicular descent. Here we show that genetic targeting of the Great gene in mice causes infertile bilateral intraabdominal cryptorchidism. The mutant gubernaculae fail to differentiate, indicating that the Great gene controls their development. Mutation screening of the human GREAT gene was performed using DHPLC analysis of the genomic DNA from 60 cryptorchid patients. Nucleotide variations in GREAT cDNA were found in both the patient and the control populations. A unique missense mutation (T222P) in the ectodomain of the GREAT receptor was identified in one of the patients. This mutant receptor fails to respond to ligand stimulation, implicating the GREAT gene in the etiology in some cases of cryptorchidism in humans.     

Increased neuronal firing in computer simulations of sodium channel mutations that cause generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+) is an autosomal dominant familial syndrome with a complex seizure phenotype. It is caused by mutations in one of 3 voltage-gated sodium channel subunit genes (SCN1B, SCN1A, and SCN2A) and the GABA(A) receptor gamma2 subunit gene (GBRG2). The biophysical characterization of 3 mutations (T875M, W1204R, and R1648H) in SCN1A, the gene encoding the CNS voltage-gated sodium channel alpha subunit Na(v)1.1, demonstrated a variety of functional effects. The T875M mutation enhanced slow inactivation, the W1204R mutation shifted the voltage dependency of activation and inactivation in the negative direction, and the R1648H mutation accelerated recovery from inactivation. To determine how these changes affect neuronal firing, we used the NEURON simulation software to design a computational model based on the experimentally determined properties of each GEFS+ mutant sodium channel and a delayed rectifier potassium channel. The model predicted that W1204R decreased the threshold, T875M increased the threshold, and R1648H did not affect the threshold for firing a single action potential. Despite the different effects on the threshold for firing a single action potential, all of the mutations resulted in an increased propensity to fire repetitive action potentials. In addition, each mutation was capable of driving repetitive firing in a mixed population of mutant and wild-type channels, consistent with the dominant nature of these mutations. These results suggest a common physiological mechanism for epileptogenesis resulting from sodium channel mutations that cause GEFS+.     

Rate-dependent changes in action potential duration and membrane currents in hamster ventricular myocytes

Hamsters are frequently studied as a model of cardiomyopathy, but the electrophysiological properties of a hamster heart are not well defined. We examined rate-dependent changes in action potentials and underlying ionic mechanisms in isolated ventricular myocytes from hamster hearts using the whole-cell configuration of the patch clamp technique. At 0.1 Hz stimulation, the mean action potential duration at 90% (APD90) and 20% (APD20) repolarization were 63+/-7 ms and 9+/-1 ms, respectively ( n=17). With increasing frequency of stimulation, APD progressively prolonged to 119+/-16 ms (APD90) and 36+/-7 ms (APD20) at 6.0 Hz. A further increase in the rate of stimulation to 8.0 Hz did not change APD significantly. Application of 4 mM 4-aminopyridine (4-AP) lengthened APD markedly and completely prevented the rate-dependent prolongation. Cd2+ (0.2 mM) shortened APD and generally attenuated the rate-dependent lengthening of APD up to 5.0 Hz, but unaffected the lengthening of APD with the further increase in the rate. At plateau voltages, there were two time-dependent currents, Ito1 and I(Ca,L). Recovery from inactivation for Ito1 had two components: t(slow)=980+/-129 ms accounting for 58% of the total fraction, and t(fast)=39+/-13 ms ( n=7). Recovery from inactivation for I(Ca,L) was rapid with t=20+/-4 ms ( n=6). Results suggest that the slow recovery from inactivation in Ito1 is the main reason for the rate-dependent prolongation of APD in hamster ventricular myocytes.