Water handling in the human distal colon in vitro: role of Na+, Cl- and HCO3-

The minute by minute net water movement (Jw) was measured, in the human distal colon in vitro, simultaneously with the transepithelial potential difference (PD) and short circuit current (SCC) with the following results: (1) An absorptive Jw (+0.36 +/- 0.04 microliters/(min.cm2)) was observed, in 21 cases, when the colon was mounted between two identical standard salines (Na+ 140, Cl- 110, HCO3- 25 mequiv./L) and in the presence of a hydrostatic pressure gradient (delta P) of 13 cm of H2O (mucosal side positive). (2) This absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (Phydr = 0.22 +/- 0.03 cm/s; Posm = 0.0020 +/- 0.005 cm/s; n = 6). (3) A fraction of this Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient while associated with a serosal side positive and partially amiloride sensitive PD (11.3 +/- 1.8 mV). (4) Both Jw and PD were dependent on the presence of Na+ in the incubating media. (5) Replacement of Cl- by SO(4)2- did not change the absorptive Jw, but increased the observed PD and the transepithelial resistance. (6) HCO3- removal strongly reduced the SCC and PD together with an important increase in Jw. Unexpectedly, other 9 colon fragments spontaneously showed a secretory Jw when mounted between two identical standard salines (-0.55 +/- 0.11 microliters/(min.cm2). In these experiments it was observed that: (7) The tissue moved water against the imposed delta P (13 cm of H2O), while the associated PD (+11.9 +/- 2.1 mV) was similar to the one observed in absorptive fragments. (8) As in the case of absorptive preparations, PD, SCC and the transport associated Jw fell to zero in the absence of Na+. (9) When SO(4)2- replaced Cl-, secretory Jw reversed to absorptive Jw, together with an increase in PD and resistance. In both absorptive and secretory preparations it was finally observed that: (10) norepinephrine (5 x 10(-6) M) decreased SCC and increased the absorptive Jw in a tightly parallel manner (half-times for each response: SCC = 11.4 +/- 2.1 min; Jw = 11.4 +/- 2.0 min, n = 4) and (11) 8-Br cyclic AMP (10(-3) M) increased SCC while simultaneously decreasing the absorptive Jw. It is concluded that the observed Jw in the distal human colon in vitro results from the complex addition of osmotic, hydrostatic and transport associated driving forces. The transport-associated Jw has absorptive and secretory components.(ABSTRACT TRUNCATED AT 400 WORDS)     

New PIN1 inhibitors identified through a pharmacophore-driven,hierarchical consensus docking strategy

PIN1 is considered as a therapeutic target for a wide variety of tumours. However, most of known inhibitors are devoid of cellular activity despite their good enzyme inhibitory profile. Hence, the lack of effective compounds for the clinic makes the identification of novel PIN1 inhibitors a hot topic in the medicinal chemistry field. In this work, we reported a virtual screening study for the identification of new promising PIN1 inhibitors. A receptor-based procedure was applied to screen different chemical databases of commercial compounds. Based on the whole workflow, two compounds were selected and biologically evaluated. Both ligands, compounds VS1 and VS2, showed a good enzyme inhibitory activity and VS2 also demonstrated a promising antitumoral activity in ovarian cancer cells. These results confirmed the reliability of our in silico protocol and provided a structurally novel ligand as a valuable starting point for the development of new PIN1 inhibitors.     

Potential of phenothiazine as a thin film dosimeter for UVA exposures.

The research reported in this paper on the changes in absorbance and the calibration of a proposed UVA (320-400 nm) dosimeter have established the phenothiazine-mylar combination as a potential UVA dosimeter for population studies of UVA exposures. The change in optical absorbance at 370 nm was employed to quantify the UVA exposures. This change starts to saturate at a change in absorbance of approximately 0.3. This relates to solar UVA exposures at a sub-tropical site on a horizontal plane of approximately three to four hours. The shape of this calibration curve varies with the season. This can be overcome in the same manner as for polysulfone where the dosimeter is calibrated for the conditions that it will be employed to measure the UVA exposures.     

Susceptibility to Heavy Metals and Cadmium Accumulation in Aerobic and Anaerobic Thermophilic Microorganisms Isolated from Deep-Sea Hydrothermal Vents

Thirty thermophilic strains isolated from heavy metal-rich hydrothermal vent sites at Lau Basin were tested for their susceptibility to cadmium, zinc, cobalt, and nickel. The 14 aerobic spore formers belonging to the genus Bacillus, 6 anaerobic fermenters from the order Thermotogales, and 10 anaerobic sulfur reducers from the order Thermococcales could be clearly distinguished according to their metal susceptibilities. The Thermococcales were found to exhibit the highest resistance to cadmium and zinc, whereas Thermotogales were highly sensitive to these metals. In contrast, the Thermotogales displayed the highest resistance to cobalt ions. No clear distinction could be established between the metal susceptibilities of these strains and seven reference organisms used for comparative studies. Cadmium resistance, slightly inducible in some cadmium-resistant bacilli, was not plasmid mediated. The amount of cadmium immobilized by the Thermotogales was related to their level of resistance to this metal. Received: 24 February 2000 / Accepted: 28 March 2000     

The narrow sheath duplicate genes: sectors of dual aneuploidy reveal ancestrally conserved gene functions during maize leaf development

The narrow sheath mutant of maize displays a leaf and plant stature phenotype controlled by the duplicate factor mutations narrow sheath1 and narrow sheath2. Mutant leaves fail to develop a lateral domain that includes the leaf margins. Genetic data are presented to show that the narrow sheath mutations map to duplicated chromosomal regions, reflecting an ancestral duplication of the maize genome. Genetic and cytogenetic evidence indicates that the original mutation at narrow sheath2 is associated with a chromosomal inversion on the long arm of chromosome 4. Meristematic sectors of dual aneuploidy were generated, producing plants genetically mosaic for NARROW SHEATH function. These mosaic plants exhibited characteristic half-plant phenotypes, in which leaves from one side of the plant were of nonmutant morphology and leaves from the opposite side were of narrow sheath mutant phenotype. The data suggest that the narrow sheath duplicate genes may perform ancestrally conserved, redundant functions in the development of a lateral domain in the maize leaf.     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

New method to measure water permeability in emptied-out Xenopus oocytes controlling conditions on both sides of the membrane

Membrane water permeability is habitually calculated from volume changes in Xenopus laevis oocytes during external osmotic challenges. Nevertheless, this approach is limited by the uncertainty on the oocyte internal composition. To circumvent this limitation a new experimental set up is introduced where the cell membrane of an emptied-out oocyte was mounted as a diaphragm between two chambers. In its final configuration the oocyte membrane was part of a closed compartment and net water movements induced swelling or shrinking of it. Volume changes were followed by video-microscopy and digitally recorded. In this manner, water movements could be continuously monitored while controlling chemical composition and hydrostatic pressure on both sides of the membrane. Using this novel experimental approach an increasing hydrostatic pressure gradient was applied to both mature (stage VI) and immature (stage IV) oocytes. The relative maximal volume change tolerated before disruption was similar in both cases (1.26+/-0.07 and 1.27+/-0.03 respectively) and similar to those previously reported under maximal osmotic stress. Nevertheless the osmotic permeability coefficient (P(OSM)) in mature oocytes ((1.72+/-0.58) x 10(-3) cm s(-1); n=6) was significantly lower than in immature oocytes ((5.18+/-0.59) x 10(-3) cm s(-1), n=5; p<0.005).     

Role of the EGF-CFC gene cripto in cell differentiation and embryo development

The EGF-CFC proteins have been recently recognized as a novel family of extracellular factors required during early vertebrate development. Cripto is the founder member of the EGF-CFC family initially related to the epidermal growth factor (EGF); its expression is increased in human colon, gastric, pancreatic and lung carcinoma and in different types of both mouse and human breast carcinomas. Genetic studies in the mouse have established an essential role of cripto in the formation and correct positioning of the anterior-posterior axis. Furthermore, the absence of cripto results in a defective precardiac mesoderm, unable to differentiate into functional cardiomyocytes. Although mouse and human Cripto have been shown to activate the ras/raf/MAP kinase pathway in mammary epithelial cell lines, genetic evidence in Zebrafish has been provided for a relationship between the EGF-CFC proteins and Nodal, a member of the transforming growth factor family. Here we review the biological role of cripto in development and differentiation, pointing out recent findings on the biochemical interactions of Cripto, Nodal and Activin-like receptors.