Demonstration of spontaneous and stretch induced urinary bladder EMG in the living rabbit

Spontaneous bladder EMG was recorded in the living rabbit from an isovolumetric bladder without chemical or electrical stimulation. Mechanical intervention, either by lifting the bladder out of the abdomen or by rapid filling, resulted in stretch induced bladder EMG. A self made epoxy resin electrode device that embedded 32 EMG recording electrodes in a matrix like pattern, each electrode Ag/AgCl, d = 0.6 mm with an interdistance of 2.3 mm, was used for registration. The recorder used a common average reference technique and a sample frequency of 400 Hz. A signal bandwidth of 0.05 to 108 Hz was available for analysis. Spontaneous EMG consisted of single spikes and bursts (2-20 spikes), but not of continuous activity. The shape of spikes was triphasic. Single spikes appeared with and without burst activity. Small (2-5 spikes) and large bursts (6-20 spikes) were discerned; small bursts not necessarily propagated across electrodes, large bursts did and were able to organize, suggesting that they were under short neuron system control. Spontaneous EMG was probably related to both contraction and relaxation. Stretch induced EMG was characterised by continuous activity on all electrodes, spikes that followed each other immediately, slowly fading away. The spikes had an elongated third phase when compared to the shape of spontaneous activity. Highest activity and amplitudes were found after lifting the bladder out of the abdomen and placing it on the electrode device. A concept is put forward in which the continuous activity is not unequivocally related to muscle shortening, but where the current stress and strain situation of the bladder tissue can cause a muscle fibre elongation upon the appearance of electrical activity. The EMG activity found was in many aspects similar to results of a previous study using mortalized rabbits. Artifact sources like the heart, respiration, or local movement between electrode and bladder could easily be identified due to the new experimental methodology used.     

Rap1 signalling: adhering to new models

Ras-like GTPases are ubiquitously expressed, evolutionarily conserved molecular switches that couple extracellular signals to various cellular responses. Rap1, the closest relative of Ras, has attracted much attention because of the possibility that it regulates Ras-mediated signalling. Rap1 is activated by extracellular signals through several regulatory proteins, and it might function in diverse processes, ranging from modulation of growth and differentiation to secretion, integrin-mediated cell adhesion and morphogenesis.     

Is it possible to replace stimulus animals by scent-filled cups in the social discrimination test?

A study in which the rat social discrimination test was refined is described. This test measures social memory by using, in general, juvenile rats as stimulus animals. Rats are offered a first juvenile to investigate (learning trial), and after a specified interval, the rats are offered the same rat and a second juvenile rat to investigate again (retrieval trial). When the rats sniff the second juvenile in the retrieval trial more than the first, social memory for the second juvenile is said to be present. This test is mainly based on scents from the juvenile. Attempts were made to refine the test to reduce the number of animals used, to enhance the scope of the test, and to improve its validity. Firstly, the stimulus animals were replaced by the scent of juveniles, in the form of cups filled with sawdust taken from cages of juvenile rats. Similar results to those in the original test were obtained when using these scents. Furthermore, male and female scents were tested, and showed the same results as for the juvenile scents. Secondly, rats were also given two cups (one scent-filled and one filled with plain sawdust) in the learning trial, to determine which allowed a more-precise delineation of motivational, discriminatory and memory components. Overall, it is possible to replace stimulus animals by scent-filled cups in the social discrimination test, to enhance the scope of the test, and to draw more-valid conclusions with respect to social memory.     

Quantification and purity determination of newly synthesized thioacridines by capillary liquid chromatography

Capillary liquid chromatography (CLC) was applied for quantification and impurity profile determination of ten newly synthesized acridine thioderivatives. A reversed-phase CLC system employing two different stationary phases, Nucleosil C18 and LiChrosorb RP-select B, was used. The mobile phase composition was optimized to get a satisfactory separation of impurities from the main acridine component in a reasonable analysis time. Significant differences in the chromatographic behavior between acridine derivatives containing and lacking amino groups were observed. Optimized separation conditions were used in CLC to measure the calibration curves of the acridine derivatives in a concentration range from 1.0 x 10(-6) to 1.0 x 10(-3) M at two different detector wavelengths (214 and 230 nm). Limits of detection and quantification of all the substances were determined. The detection limits went down to units of microM for most of the derivatives. CLC was also demonstrated to be a suitable method for the purity determination of test batches of the acridine thioderivatives.     

Structure and regulation of the cAMP-binding domains of Epac2

Cyclic adenosine monophosphate (cAMP) is a universal second messenger that, in eukaryotes, was believed to act only on cAMP-dependent protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Recently, guanine nucleotide exchange factors specific for the small GTP-binding proteins Rap1 and Rap2 (Epacs) were described, which are also activated directly by cAMP. Here, we have determined the three-dimensional structure of the regulatory domain of Epac2, which consists of two cyclic nucleotide monophosphate (cNMP)-binding domains and one DEP (Dishevelled, Egl, Pleckstrin) domain. This is the first structure of a cNMP-binding domain in the absence of ligand, and comparison with previous structures, sequence alignment and biochemical experiments allow us to delineate a mechanism for cyclic nucleotide-mediated conformational change and activation that is most likely conserved for all cNMP-regulated proteins. We identify a hinge region that couples cAMP binding to a conformational change of the C-terminal regions. Mutations in the hinge of Epac can uncouple cAMP binding from its exchange activity.     

Contribution of plasma proteins to splanchnic and total anabolic utilization of dietary nitrogen in humans

Splanchnic tissues are largely involved in the postprandial utilization of dietary amino acids, but little is yet known, particularly in humans, about the relative contributions of different splanchnic protein pools to splanchnic and total postprandial anabolism. Our aim was to develop a compartmental model that could distinguish dietary nitrogen (N) incorporation among splanchnic constitutive, plasma (splanchnic exported), and peripheral proteins after a mixed-protein meal in humans. Eight healthy subjects were fed a single mixed meal containing 15N-labeled soy protein, and dietary N postprandial kinetics were measured in plasma free amino acids, proteins, and urea and urinary urea and ammonia. These experimental data and others previously obtained for dietary N kinetics in ileal effluents under similar experimental conditions were used to develop the compartmental model. Six hours after the mixed-meal ingestion, 31.5, 7.5, and 21% of ingested N were predicted to be incorporated into splanchnic constitutive, splanchnic exported, and peripheral proteins, respectively. The contribution of splanchnic exported proteins to total splanchnic anabolism from dietary N was predicted to be approximately 19% and to remain steady throughout the simulation period. Model behavior and its predictions were strongly in line with current knowledge of the system and the scarce, specific data available in the literature. This model provides the first data concerning the anabolism of splanchnic constitutive proteins in the nonsteady postprandial state in humans. By use of only slightly invasive techniques, this model could help to assess how the splanchnic anabolism is modulated under different nutritional or pathophysiological conditions in humans.     

Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs

Epac1 (cAMP-GEFI) and Epac2 (cAMP-GEFII) are closely related guanine nucleotide exchange factors (GEFs) for the small GTPase Rap1, which are directly regulated by cAMP. Here we show that both GEFs efficiently activate Rap2 as well. A third member of the family, Repac (GFR), which lacks the cAMP dependent regulatory sequences, is a constitutive activator of both Rap1 and Rap2. In contrast to Epac1, Epac2 contains a second cAMP binding domain at the N terminus, as does the Epac homologue from Caenorhabditis elegans. Affinity measurements show that this distal cAMP binding domain (the A-site) binds cAMP with much lower affinity than the cAMP binding domain proximal to the catalytic domain (the B-site), which is present in both Epac1 and Epac2. Deletion mutant analysis shows that the high affinity cAMP binding domains are sufficient to regulate the GEFs in vitro. Interestingly, isolated fragments containing the B-sites of either Epac1 or Epac2, but not the A-site from Epac2, inhibit the catalytic domains in trans. This inhibition is relieved by the addition of cAMP. In addition to the cAMP binding domains, both Epac1 and Epac2 have a DEP domain. Deletion of this domain does not affect regulation of Epac1 activity but affects membrane localization. From these results, we conclude that all three members of the Epac family regulate both Rap1 and Rap2. Furthermore, we conclude that the catalytic activity of Epac1 is constrained by a direct interaction between GEF and high affinity cAMP binding domains in the absence of cAMP. Epac1 becomes activated by a release of this inhibition when cAMP is bound.     

RalGEF2, a pleckstrin homology domain containing guanine nucleotide exchange factor for Ral

Ral is a ubiquitously expressed Ras-like small GTPase. Several guanine nucleotide exchange factors for Ral have been identified, including members of the RalGDS family, which exhibit a Ras binding domain and are regulated by binding to RasGTP. Here we describe a novel type of RalGEF, RalGEF2. This guanine nucleotide exchange factor has a characteristic Cdc25-like catalytic domain at the N terminus and a pleckstrin homology (PH) domain at the C terminus. RalGEF2 is able to activate Ral both in vivo and in vitro. Deletion of the PH domain results in an increased cytoplasmic localization of the protein and a corresponding reduction in activity in vivo, suggesting that the PH domain functions as a membrane anchor necessary for optimal activity in vivo.     

Retention of bacteria on a substratum surface with micro-patterned hydrophobicity

Bacteria adhere to almost any surface, despite continuing arguments about the importance of physico-chemical properties of substratum surfaces, such as hydrophobicity and charge in biofilm formation. Nevertheless, in vivo biofilm formation on teeth and also on voice prostheses in laryngectomized patients is less on hydrophobic than on hydrophilic surfaces. With the aid of micro-patterned surfaces consisting of 10-microm wide hydrophobic lines separated by 20-microm wide hydrophilic spacings, we demonstrate here, for the first time in one and the same experiment, that bacteria do not have a strong preference for adhesion to hydrophobic or hydrophilic surfaces. Upon challenging the adhering bacteria, after deposition in a parallel plate flow chamber, with a high detachment force, however, bacteria were easily wiped-off hydrophobic lines, most notably when these lines were oriented parallel to the direction of flow. Adhering bacteria detached slightly less from the hydrophilic spacings in between, but preferentially accumulated adhering on the hydrophilic regions close to the interface between the hydrophilic spacings and hydrophobic lines. It is concluded that substratum hydrophobicity is a major determinant of bacterial retention while it hardly influences bacterial adhesion.     

Copurification of P6, MRP8, and MRP14 from Human Granulocytes and Separation of Individual Proteins

A method is described for purification of P6, MRP8, and MRP14, three calcium-binding proteins assigned to the S100 protein family. The purification procedure included preparation of human granulocytes, ammonium sulfate precipitation, and anion-exchange chromatography and resulted in the copurification of P6, MRP8, and MRP14. Individual proteins were separated by either preparative isoelectric focusing or preparative SDS–PAGE. The procedure was carried out in the course of 4 days and yielded several milligrams of essentially pure P6, MRP8, and MRP14 in either native or denatured form.