Pathways for organic osmolyte synthesis in rabbit renal papillary tissue, a metabolic study using 13C-labeled substrates

Renal papillary collecting duct cells have been postulated to adapt their intracellular osmolality to the large changes in interstitial osmolality by changing their content of 'non-perturbing' organic osmolytes such as sorbitol and myo-inositol. 13C-NMR was used in this study to elucidate the metabolic pathways leading to a synthesis of those compounds. Incubation of rabbit renal papillary tissue with [1-13C]glucose showed label scrambling mainly into sorbitol (C-1) and lactate (C-3). This result confirms activity of aldose reductase and glycolytic enzymes in renal papillary cells. Using [3-13C]alanine or [2-13C]pyruvate as carbon source, 13C-labeling of sorbitol and myo-inositol was observed, indicating that renal papillary tissue possesses, in addition, gluconeogenic activity. The latter assumption is supported by the result that in enzyme assays rabbit kidney papilla and isolated rat kidney papillary collecting duct cells show significant fructose-1,6-bisphosphatase activity.     

Green-backed herons (Butorides striatus) possibly using a lure and using apparent bait

Zusammenfassung In Burkina Faso ließ ein Mangrovereiher offensichtlich gezielt eine Asclepiadaceen-Blüte aus 20 cm Höhe auf die Wasseroberfläche fallen und verharrte danach einige Sekunden mit halb-gestrecktem Hals. Bei einer weiteren Beobachtung in Niger plazierte ein Mangrovereiher einen kleinen Gegenstand auf der Wasseroberfläche. Bevor der Gegenstand mit dem Wind außer Reichweite trieb, holte ihn der Reiher und legte ihn auf der Luvseite wieder auf der Wasseroberfläche ab. Der Vorgang wiederholte sich mehrere Male, dabei gelang es dem Reiher, einen Fisch zu erbeuten, der allerdings wieder entkam. Anderntags setzte an derselben Stelle ein Mangrovereiher in gleicher Weise offensichtlich einen Käfer ein. Ähnliche Beobachtungen werden kurz diskutiert.     

Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.     

Phenylethanolamine N-methyltransferase inhibition: re-evaluation of kinetic data

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we demonstrate that a routinely used assay for screening PNMT inhibitors is not appropriate for those inhibitors having K(i) values less than 1 microM. A revised assay has been developed that shows some inhibitors bind two orders of magnitude more tightly than previously reported.     

Getting the adrenaline going: crystal structure of the adrenaline-synthesizing enzyme PNMT

BACKGROUND: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. RESULTS: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 A. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. CONCLUSIONS: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.     

Enrichment of non–apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting

Cryopreservation increases the rate of apoptotic spermatozoa withdecreased capability to fertilise oocytes. In order to optimise thefertilisation rates, especially in assisted reproduction the use of apoptoticsperms should be avoided. Early events of apoptosis in cryopreservedspermatozoaare not detectable by conventional methods. However, the surface of apoptoticspermatozoa is characterised by externalisation of phosphatidylserine (PS),which has a high affinity to Annexin V. Therefore, colloid paramagneticAnnexin-V-conjugated microbeads (AN-MB) were tested fortheir ability to eliminate apoptotic spermatozoa from a total of 40 fresh andinTEST yolk buffer cryopreserved semen samples which were provided by 15 healthyvolunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) thesperm suspensions were divided into 2 sperm fractions depending on boundmagnetic Annexin V-microbeads (AN-MB) to spermatozoa. Asadditional markers of apoptosis CD95 (Fas, APO-1) on the sperm surfaceand activated caspases in the cytosol were detected in both fractions.Supplementary investigations comprised eosin-supravital staining andcomputer assisted sperm motion analysis. The separation was supervised by flowcytometric analysis of spermatozoa labelled with FITC-conjugated antiAnnexin V-antibodies.Analyses of the magnetic inactive sperm fraction (AN-MB-negative)showed CD95 on 0.6 ± 0.3% (X ± SEM) of spermatozoa andonly3.2 ± 0.5% were stainable with eosin, whereas, 40.6 ±6.7% of the remaining cells in the column appeared to be CD95 positiveand 99.8 ± 0.1% stainable with eosin after cryopreservation.Indeed the overall amount of CD95 positive spermatozoa did not significantlyincrease after cryopreservation (2.5 ± 0.5% vs. 4.3 ±1.2%; p > 0.05). Activated caspases were found in 21.8 ±2.6% of the spermatozoa in fresh and in 47.7 ± 5.8% ofcryopreserved semen samples (p < 0.01). The separation procedure of thecryopreserved spermatozoa reduced significantly the quantity of thosecontainingactivated caspases to 9.3 ± 2.2% within theAN-MB-negative fraction. In contrast 89.1 ± 2.3% ofAN-MB-positive sperms showed activation of these proteolyticenzymes. Flow cytometric analyses using FITC-conjugated anti AnnexinV-antibodies for monitoring of AN-MB-binding to spermatozoashowed 5.2 ± 1.0% labelled spermatozoa in the AN-MBnegative fraction and 72.6 ± 2.7% labelled spermatozoa in theAN-MB positive one. There was no significant influence of the separationcolumn and the magnetic field on the sperm functions. The passage through thecolumn led to a sperm loss of 0.8 ± 1.2%.Conclusion: The binding of paramagnetic AnnexinV-conjugated microbeads is an excellent method to eliminate spermatozoaat early apoptotic stages from cryopreserved semen samples. A deleteriousinfluence of the separation column and the magnetic field on the spermatozoawasnot observed.     

A GFP-based system to uncouple mRNA transport from translation in a single living neuron

An inducible fluorescent system based on GFP is presented that allows for the uncoupling of dendritic mRNA transport from subsequent protein synthesis at the single cell level. The iron-responsive element (IRE) derived from ferritin mRNA in the 5'-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron. The addition of the full-length 3'-UTR of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) after the stop codon of the GFP reading frame targets the reporter mRNA to dendrites of transfected fully polarized hippocampal neurons. As we show by time-lapse videomicroscopy, iron specifically turns on GFP reporter protein synthesis in a single transfected hippocampal neuron. We investigate whether GFP expression is affected--in addition to iron--by synaptic activity. Interestingly, synaptic activity has a clear stimulatory effect. Most importantly, however, this activity-dependent protein synthesis is critically dependent on the presence of the full-length 3'-UTR of CaMKIIalpha confirming that this sequence contains translational activation signals. The IRE-based system represents a new convenient tool to study local protein synthesis in mammalian cells where mRNA localization to a specific intracellular compartment occurs.     

Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNA^Lys _UUU, tRNA^Glu _UUC and tRNA^Gln _UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm⁵s²U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm⁵ side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm⁵s²U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm⁵U and s²U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm⁵s²U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA^Lys _UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm⁵ and s² wobble uridine modification that could account for growth impairment and mainly originates from tRNA^Lys _UUU hypomodification and malfunction.     

The tify family previously known as ZIM

The ZIM domain was originally identified in the ZIM protein (BAA97679; Zinc-finger protein expressed in Inflorescence Meristem). Since then it has been found in other proteins and the corresponding genes have been grouped into a plant-specific family. However, the family lacks consistency in its classification among different databases. Here, we try to clarify this incongruity by presenting an overview of the Arabidopsis proteins having this domain. The presented genome-wide survey can be seen as a start point to reveal the unknown function of these proteins. Furthermore, because of the confusing ZIM nomenclature being used at present, we propose to rename the domain and family as tify, after the most conserved amino acid motif characterizing the members of this family.