Small molecule absorption by PDMS in the context of drug response bioassays.

The polymer polydimethylsiloxane (PDMS) is widely used to build microfluidic devices compatible with cell culture. Whilst convenient in manufacture, PDMS has the disadvantage that it can absorb small molecules such as drugs. In microfluidic devices like "Organs-on-Chip", designed to examine cell behavior and test the effects of drugs, this might impact drug bioavailability. Here we developed an assay to compare the absorption of a test set of four cardiac drugs by PDMS based on measuring the residual non-absorbed compound by High Pressure Liquid Chromatography (HPLC). We showed that absorption was variable and time dependent and not determined exclusively by hydrophobicity as claimed previously. We demonstrated that two commercially available lipophilic coatings and the presence of cells affected absorption. The use of lipophilic coatings may be useful in preventing small molecule absorption by PDMS.

Phosphine-imine and -enamido ligands for acceptorless dehydrogenation catalysis.

A highly tunable phosphine-imine ligand family is introduced. Following metallation with ruthenium, deprotonation of the ligand affords a phosphine-enamido species. Complexes with the ligand in both the imine and enamido forms are active toward acceptorless dehydrogenation reactions.

Maternal undernutrition during early to midgestation programs tissue-specific alterations in the expression of the glucocorticoid receptor, 11beta-hydroxysteroid dehydrogenase isoforms, and type 1 angiotensin ii receptor in neonatal sheep.

We have investigated the effects of maternal nutrient restriction in the sheep during the period of rapid placental growth (i.e. 28-77 days gestation; term = 147 days) on feto-placental growth and expression of the glucocorticoid receptor (GR), types 1 and 2 11beta-hydroxysteroid dehydrogenase (11betaHSD1, 11betaHSD2), and types 1 and 2 angiotensin II receptor (AT1, AT2) in fetal and neonatal offspring. Ewes (n = 63) of similar age, body weight, and body composition were randomly allocated to a nutrient-restricted (NR) group in which they consumed 3.2 MJ/day metabolizable energy (ME; equivalent to 50% of predicted requirements) or to a control group in which they consumed 6.7 MJ/day ME (equivalent to 110% of predicted requirements). After 77 days gestation, ewes from both dietary groups consumed close to 100% of ME requirements up to term. Newborn offspring of NR ewes were of similar body weight, but had increased crown-rump length, greater placental weight, and increased placental/body weight ratio (P < 0.01) compared with controls. Their kidneys were heavier (P < 0.05), but shorter in length, with increased ratios of transverse width to length (P < 0.001). GR messenger RNA (mRNA) expression in neonatal offspring from NR ewes was increased in adrenal, kidney, liver, lung, and perirenal adipose tissue (P < 0.01). Conversely, 11betaHSD1 mRNA expression was unaffected, except in perirenal adipose tissue, where it was higher in lambs born to NR ewes (P < 0.01). 11betaHSD2 mRNA expression was decreased in adrenals and kidney (P < 0.001). Maternal NR also resulted in significantly increased AT1 expression in those tissues in which expression of GR was increased and/or 11betaHSD2 was decreased, i.e. adrenals, kidney, liver, and lung. AT2 expression was unaffected by maternal NR. Although 11betaHSD2 mRNA was undetectable in term placenta, it was abundant in midgestation placenta and was lower after maternal NR (P < 0.001). There was close agreement between levels of 11betaHSD enzyme (i.e. 11beta-dehydrogenase and 11-oxoreductase) activities and abundance of 11betaHSD1 mRNA and 11betaHSD2 mRNA expression. The persistence of tissue-specific increases in the expression of GR, 11betaHSD1 and AT1 and decreases in the expression of 11betaHSD2 in adrenals and kidney in newborn offspring in response to a defined period of maternal nutrient restriction during early to midgestation suggests that gene expression has been programmed by nutrient availability to the fetus before birth. These data suggest key potential mechanisms by which maternal nutrition prenatally programs physiological pathways, such as the renin-angiotensin system, in the offspring that may lead to raised blood pressure and other cardiovascular disease risk factors in later life.

Electroporation of peptides into adherent cells in situ.

To study the effect of protein interactions in vivo upon cellular functions,such complexes may be disrupted through the introduction of peptides corresponding to the proteins' points of contact. In this communication a simple, rapid and reproducible procedure for peptide introduction into adherent cells by electroporation is described. Cells are grown on electrically conductive, optically transparent indium-tin oxide at the time of pulse delivery. Several electrode and slide configurations, necessary to obtain non-electroporated cells adjacent to the electroporated ones as a control, are outlined. Careful control of electric field strength achieved the introduction of the peptide into essentially 100% of the cells while this treatment caused no detectable disruption of their division cycle.

Electroporation of adherent cells in situ.

A simple, rapid, and reproducible procedure for the introduction of macromolecules into adherent mammalian cells by electroporation is described. Cells were growing on a glass surface coated with electrically conductive, optically transparent indium-tin oxide at the time of pulse delivery. Several factors affected the optimal voltage for permeation of a given line including the metabolic state of the cells and their degree of spreading onto the conductive growth surface. Careful control of the electric field strength resulted in almost 100% of the cells containing introduced antibodies without any detectable change in the length of their division cycle. Higher voltages were required for the stable expression of DNA than for the introduction of antibodies, resulting in a significant rate of cell death.

A novel technique for the study of Ras activity: electroporation of [alpha-32P]GTP.

An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [alpha-32P]GTP was introduced by in situ electroporation into mouse C3H10T1/2 fibroblasts or their ras(val12)-transformed derivatives. After a 3-hr incubation at 37 degrees C, Ras was immunoprecipitated from cell extracts and the Ras-bound GTP/GTP + GDP ratio was determined by thin-layer chromatography. Contrary to Streptolysin-O permeabilization, the cells are not affected in any detectable way by the procedure, so that [alpha-32P]GTP binding and conversion to [alpha-32P]GDP can be studied over a period of time for the measurement of steady-state Ras activity. The results show that careful control of electric field intensity results in a great increase in the efficiency and specificity of labelling compared to the addition of [32P]orthophosphate to the culture medium, while the GTP/GTP + GDP ratios obtained were essentially the same as after in vivo labeling.

A novel technique for the study of intercellular, junctional communication: electroporation of adherent cells on a partly conductive slide.

One of the effects of neoplastic transformation by a variety of factors is a decrease in gap junctional, intercellular communication (GJIC). The investigation of junctional permeability is usually conducted through the microinjection of the fluorescent dye, Lucifer yellow, followed by observation of its migration into neighboring cells. This is a time-consuming approach, requiring expensive equipment. To overcome these problems, a novel technique was devised which takes advantage of the ability of short electric pulses to create transient "pores" on the cell membrane through which Lucifer yellow can enter, simultaneously and into large numbers of cells, with minimal disturbance to cellular metabolism. Cells were grown on a glass slide, half of which was coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse was applied in the presence of Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area was microscopically observed under fluorescence illumination. Using this technique, we investigated the relationship between expression of the middle tumor antigen of polyoma virus (mT) and GJIC in two representative cell systems with different responses to mT. The results show that low mT expression levels, although unable to transform rat F111 cells fully, are able to interrupt GJIC. Although parts of this mechanism might be mediated through protein kinase C (PKC), mT appears to have additional functions. PKC, however, had the opposite effect upon junctional permeability in a clone of mouse NIH-3T3 fibroblasts; intercellular communication in these cells appears to require PKC activity.

Applications of electroporation of adherent cells in situ, on a partly conductive slide.

Nontraumatic, simple, and reproducible procedures for the introduction of nonpermeant molecules into adherent mammalian cells by in situ electroporation are described. Cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse is applied in the presence of the molecules to be introduced, and their effect on the cellular phenotype can be observed. The cells growing on the nonconductive side of the slide do not receive any pulse and serve as controls. Careful adjustment of electric field strength can achieve the introduction of the molecules into essentially 100% of the cells, and this treatment causes no detectable disruption to cellular metabolism. This is applied in the presence of the fluorescent dye, Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide. The migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination.

Influence of relative size at birth on growth and glucose homeostasis in twin lambs during juvenile life.

The effect of differences in size at birth on growth and glucose homeostasis between female twin lambs during juvenile life was examined. Twenty-six sets of twins were entered into the study, of which ten were used for organ sampling at birth and 16 were studied over the first year of life. Eleven sets were defined as being mismatched for birthweight as the weight difference between twins was >25%, with light lambs weighing 4.1 +/- 0.3 kg and heavy lambs weighing 5.1 +/- 0.1 kg. All remaining twins were matched in bodyweight, weighing 4.6 +/- 0.5 kg. During the rapid period of juvenile growth (i.e. one, three and six months of age) and following stabilization of bodyweight (i.e. 12 months of age) glucose tolerance tests were performed by intravenously injecting 0.8 mg kg(-1) bodyweight glucose. This was followed the next day with an insulin tolerance test, performed by intravenously injecting 0.08 units kg(-1) bodyweight insulin. At birth there were no differences in organ weight as a fraction of total bodyweight between matched and mismatched twins, but the ratio of liver to brain weight was lower in light compared with heavy twins. Light lambs remained lighter than their twins up to six months of age, and crown-rump length was also shorter. At one and three months of age there were no differences in basal plasma glucose concentrations between the groups, but glucose tolerance was greater in light compared with heavy lambs at one and six months of age. Insulin tolerance was greater in light compared with matched lambs at one and six months of age. In conclusion, it has been shown that size at birth of one twin in relation to its co-twin is one factor determining glucose regulation during postnatal life. This not only affects glucose and insulin tolerance but also growth over the first six months of age.

Changes in hydrolytic enzyme activities of naïve Atlantic salmon Salmo salar skin mucus due to infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation.

The changes in the activities of mucus hydrolytic enzymes and plasma cortisol levels were examined following infection of Atlantic salmon Salmo salar with the salmon louse Lepeophtheirus salmonis and these changes were compared with those resulting from elevated plasma cortisol. Salmon were infected at high (Trial 1; 178 +/- 67) and low (Trial 2; 20 +/- 13) numbers of lice per fish and the activities of proteases, alkaline phosphatase, esterase and lysozyme in the mucus, as well as plasma cortisol levels were determined. At both levels of infection, there were significant increases of protease activity over time (1-way K-WANOVA; Trial 1, p = 0.004; Trial 2, p < 0.001). On several sampling days, generally on later days in the infections, the mucus protease activities of infected fish were significantly higher than control fish (Student's t-tests; p < 0.05). In addition, zymography experiments demonstrated bands of proteases at 17 to 22 kDa in the mucus of infected salmon that were absent in the mucus from non-infected fish and absent in the plasma of salmon. The intensity of these protease bands increased in the mucus over the course of both infections. However, plasma cortisol levels were elevated only in the heavily infected fish from the first trial. At high infection levels (Trial 1), alkaline phosphatase activity was higher in the mucus of infected fish at all days (t-test, p < 0.05). However, at the lower infection level (Trial 2), the mucus alkaline phosphatase activity did not differ significantly between infected and non-infected fish. Esterase and lysozyme activities were very low and did not change with time nor between non-infected and infected salmon in either challenge. Mucus enzyme activities of cortisol-implanted salmon did not change over time, nor were there any differences in activities between cortisol-implanted and control salmon. The present study demonstrates biochemical changes resulting from sea lice infection of Atlantic salmon occurring at the site of host-pathogen interaction, the mucus layer. However, the origin of these enzymes, whether host or pathogen, remains to be determined.

Characterization of proteases in the skin mucus of Atlantic salmon (Salmo salar) infected with the salmon louse (Lepeophtheirus salmonis) and in whole-body louse homogenate.

As part of an investigation of the biochemical interactions between the salmon louse Lepeophtheirus salmonis and Atlantic salmon Salmo salar, we characterized protease activity in the skin mucus of noninfected Atlantic salmon and Atlantic salmon infected with L. salmonis and in an L. salmonis whole-body homogenate. Zymography revealed that mucus from infected salmon contained a series of low-molecular-mass (17-22 kDa) serine proteases that were not present in the mucus of noninfected salmon. Based on molecular mass, inhibition studies, and affinity chromatography, the series of proteases was identified as being trypsin-like. Similar proteases were observed in the L. salmonis homogenate and in mucus from noninfected Atlantic salmon following a 1-hr incubation with live L. salmonis. An antibody raised against Atlantic salmon trypsin failed to recognize any proteases in the mucus of noninfected salmon or infected salmon or in the L. salmonis homogenate. Collectively, these findings suggest that the trypsin-like proteases present in the mucus of infected Atlantic salmon were produced by L. salmonis, possibly to aid in feeding and evasion of host immune responses.

Baroreflex responses to the stress of severe hemorrhage in the rat.

In two groups of anesthetized (sodium pentobarbital), mature Sprague-Dawley rats, 1) aged 2 years and weighing 300-400 grams, 2) aged 6 months weighing 200-300 grams, baroreflex-induced circulatory responses to pressor (graded doses phenylephrine) and depressor (graded doses nitroglycerine) agents were compared to those occurring during progressive hemorrhage in the same animals. Graded withdrawals of blood from the femoral artery elicited progressive hypotension accompanied by bradycardia rather than expected tachycardia. Graded doses of phenylephrine (2.5 ug to 40 ug bolus, via femoral vein) regularly induced elevations in arterial blood pressure with associated reflex bradycardia. Similarly graded doses of nitroglycerine induced a marked decline in arterial blood pressure, without expected tachycardia. As hypotension became more severe (during hemorrhage), atrioventricular conduction slowed and A-V block developed, resulting in statistically greater slowing in ventricular than in atrial excitation and contractile cycles. Heart failure during hemorrhage in the rat is characterized sequentially by severe bradycardia, depressed atrial contractile force, impaired conduction and A-V block, terminating in ventricular, atrial, and finally, in pacemaker failure. Baroreceptor reflexes were blunted or even absent in both young and old animals during induced hypotension.

Developing nurse leaders of the future.

Nurse leaders need to combine clinical excellence with the ability to harmonise staff's personal goals, the professional agenda and the goals of the organisation.

Human alpha- and beta-calcitonin gene-related peptides are vasodilators in human chorionic plate vasculature.

Calcitonin gene-related peptide receptors are known to be present in the placenta. We studied the effect of alpha- or beta-calcitonin gene-related peptide or carrier on human chorionic plate arteries preconstricted with angiotensin II. Both forms of calcitonin gene-related peptides evoked dose-dependent relaxation, the magnitude of which was proportional to the initial response to angiotensin II.

Growth on indium-tin oxide-coated glass enhances 32P-phosphate uptake and protein labelling of adherent cells.

A method to improve the efficiency of labelling of adherent cells with radioactive 32p is described. Cells are grown on a glass surface which is coated with indium-tin oxide, a commercially available, transparent material which permits excellent cell adhesion and growth. The results show that a 2 to 3-fold increase in 32p uptake by the cells can be achieved by growing cells on this material, compared to conventional tissue culture plastic.

Influence of route of delivery and ambient temperature on thermoregulation in newborn lambs.

We examined the effect of route of delivery on brown adipose tissue (BAT) function and thermoregulation in lambs born either vaginally at term or by cesarean section close to term. Immediately after birth, lambs were placed in a warm (30 degrees C; WD) or cool (15 degrees C; CD) ambient temperature, and measurements of colonic temperature, plus heat production (i.e., oxygen consumption and carbon dioxide production), were recorded for 6 h. Over the first 30 min of life, colonic temperature remained constant in vaginally delivered lambs and was lower in the WD group. Following cesarean section delivery, colonic temperature declined rapidly, a response that was greater in the CD group. Cesarean section-delivered lambs had an increased reliance on shivering thermogenesis and restored colonic temperature after 2 h, and by 6 h these parameters were higher than in lambs born vaginally. Irrespective of delivery, temperature, plasma thyroid hormone concentrations and norepinephrine content of BAT were lower in lambs born by cesarean section compared with those born vaginally. Plasma cortisol concentrations and epinephrine content of BAT were greater in lambs born by cesarean section. The amount of uncoupling protein and level of guanosine 5'-diphosphate binding in BAT were higher in vaginally delivered than in cesarean section-delivered lambs, and for each group mean values were greater for CD than WD lambs. Cesarean section delivery results in altered thyroidal, adrenal, and sympathetic activity, which appears to have a marked influence on BAT function, thereby contributing to distinct differences in thermoregulation compared with lambs born vaginally.

Short circuit current in rat adrenal capsule/glomerulosa.

Angiotensin II (AII) and potassium are the primary regulators of aldosterone secretion by adrenal zona glomerulosa cells. Electrophysiological studies using isolated adrenal tissues and dispersed zona glomerulosa cells show that stimulation by these secretagogues results in depolarisation of the plasma membrane and the opening of voltage-sensitive ion channels. The concept that these cells act together to create a polarity within the gland has not been examined. Whole adrenal capsule/glomerulosa preparations were studied in Ussings chambers. An increase in [KCl] to either side of the capsule resulted in a concentration-related change in short-circuit current (SCC). KCl added externally caused an increase in SCC, indicating net inward movement of positive ions or net outward movement of negative ions. Internal KCl had a smaller opposite effect. Use of the non-specific potassium channel blocker tetraethylammonium chloride (TEA) resulted in an increase in SCC regardless of which side the addition was made, although on occasions the responses to TEA and internal KCl were unexpectedly reversed. The aldosterone antagonist spironolactone produced a variable change in SCC suggesting an autocrine/paracrine role for aldosterone on the adrenal cortex. No responses were observed with the addition of AII, ACTH or aldosterone, though these may be present in excess. The results suggest that ion gradients may be created by stimulation that conceivably have a role in cellular organisation.