EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.     

Physiologically relevant one-compartment pharmacokinetic models for skin. 1. Development of models

Many studies have used pharmacokinetic (compartment) models for skin to predict or analyze absorption of chemicals through skin. In these studies, several different definitions of the rate constants were used. The purpose of this study was to develop a general procedure for relating compartment model rate constants to dermal absorption parameters, such as permeability and partition coefficients, and to assess whether different definitions of the rate constants produce different results. Rate constant expressions were developed by requiring a one-compartment model to match a one-membrane model at specific conditions. Because a membrane model contains more information than a compartment model, a compartment model cannot match the membrane model in all respects. Consequently, many compartment models (i.e., different definitions of the rate constants) can be developed which match the membrane model for different conditions. Using this procedure, 11 different compartment models were developed and compared to the membrane model for four different dermal absorption scenarios. The compartment model that most closely matches the membrane model depends on the specific exposure scenario and what is to be predicted. One of the new compartment models agrees reasonably well with the membrane model, for the cases considered.     

Novel diffusion cell for in vitro transdermal permeation, compatible with automated dynamic sampling

The development of a new diffusion cell for in vitro transdermal permeation is described. The so-called Kelder cells were used in combination with the ASPEC system (Automatic Sample Preparation with Extraction Columns), which is designed for the automation of solid-extractions (SPE). Instead of SPE columns, 20 Kelder cells were placed in the racks. This allowed automatic sampling of up to 20 cells for 24 h in a dynamic mode. The cells consist of an inlet compartment, a donor compartment and a receptor compartment. The size and the depth of the inlet compartment were important to avoid entrapment of air bubbles in the receptor compartment. The Kelder cells mimic blood flow beneath the skin by replacement of the permeating drug every 2 min. Hence sink condition are more easily maintained than with the static Franz diffusion cell. The performance of the cells was tested with permeation experiments using atropine as a model drug permeating through an artificial membrane (Silastic). The use of this skin model minimized the variability in permeation of atropine as compared with human skin.     

The distribution of SMN protein complex in human fetal tissues and its alteration in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of motor neurons of the spinal cord and muscular atrophy. SMA is caused by alterations to the survival of motor neuron (SMN) gene, the function of which has hitherto been unclear. Here, we present immunoblot analyses showing that normal SMN protein expression undergoes a marked decay in the postnatal period compared with fetal development. Morphological and immunohistochemical analyses of the SMN protein in human fetal tissues showed a general distribution in the cytoplasm, except in muscle cells, where SMN protein was immunolocalized to large cytoplasmic dot-like structures and was tightly associated with membrane-free heavy sedimenting complexes. These cytoplasmic structures were similar in size to gem. The SMN protein was markedly deficient in tissues derived from type I SMA fetuses, including skeletal muscles and, as previously shown, spinal cord. While our data do not help decide whether SMA results from impaired SMN expression in spinal cord, skeletal muscle or both, they suggest a requirement for SMN protein during embryo-fetal development.     

The effect of endothelin and its antagonist Bosentan on hemodynamics and microvascular exchange in cirrhotic rat liver

BACKGROUND/AIMS: To characterize the effects of endothelin-1 and of Bosentan, a mixed endothelin antagonist, on hepatic hemodynamics in cirrhotic animals in vivo and on hepatic microvascular exchange in the perfused rat liver. METHODS: Biliary cirrhosis was induced by bile duct ligation, and micronodular cirrhosis by chronic exposure to phenobarbital/CCl4 in male rats. Hepatic hemodynamics were studied under basal conditions and after administration of Bosentan (3-30 mg/kg) by the microsphere technique. Microvascular exchange was assessed in the in situ perfused rat liver by the multiple indicator dilution technique. RESULTS: Bosentan lowered portal pressure in a dose-dependent fashion; at the highest dose tested, this decrease averaged -29+/-11 and -26+/-8% in biliary and micronodular cirrhosis, respectively (p<0.01). This was achieved mainly via a marked decrease in hepatic arterial flow. In the perfused liver, endothelin-1 induced a dose-dependent vasoconstriction; up to 10(-9) mol/l; this was not associated with any effect on viability. At this dose, endothelin-1 markedly decreased extravascular albumin space in both controls and micronodular cirrhosis; this could be antagonized by Bosentan 10(-5) mol/l. CONCLUSIONS: Endothelin-1 affects hepatic microvascular exchange, presumably by a direct effect on hepatic sinusoidal endothelial cells. A mixed endothelin antagonist lowers portal pressure in vivo, presumably by acting on hepatic stellate cells, and counteracts the microvascular effects of endothelin-1 in vitro. These properties could prove useful in treatment of portal hypertension.     

Differential acetylcholine and GABA release from cultured chick retina cells

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.     

Synthesis and analgesic properties of new 5-thioaryl pyrazole derivatives

A new series 5-thio aryl pyrazole derivatives were proposed aiming analgesic activity. In this work, 8 new compounds of this class were synthesized using usual synthetic methodology, having as key intermediate the 3-methyl-4-nitro-5-chloropyrazole-1-phenyl derivative and subsequent reaction with several nucleophiles sulfides. Pharmacological evaluation of this series showed analgesic activity in the some extent in especially for 5-(4-bromophenyl)-thio-3-methyl-4-nitro-1-phenylpyrazole which was the most potent in this series, presenting an analgesic action comparable to that show by dipyrone.