Cyclic and linear vasopressin V1 and V1/V2 antagonists containing arginine in the 4-position

Summary Substitution of arginine for glutamine in the 4-position of a vasopressin V₁ antagonist has been reported to turn it into an agonist. We resynthesized this 4-arginine analog and synthesized additional cyclic and linear vasopressin antagonists containing a 4-arginine. The presence of a 4-arginine in the resynthesized and new analogs had relatively minor effects on their antivasopressin V₁ and V₂ antagonistic potencies.     

Vasopressin antagonists

Effects of vasopressin via V1a- and V2-receptors are closely implicated in a variety of water-retaining diseases and cardiovascular diseases, including heart failure, hyponatraemia, hypertension, renal diseases, syndrome of inappropriate antidiuretic hormone secretion, cirrhosis and ocular hypertension. As vasopressin receptors are found in many different tissues, vasopressin antagonists may benefit the treatment of disorders such as cerebral ischaemia and stroke, Raynaud’s disease, dysmenorrhoea and tocolytic treatment. V1b selective vasopressin antagonists are discussed in terms of their usefulness in the treatment of emotional and psychiatric disorders. The vaptans are vasopressin receptor antagonists with V1a (relcovaptan) or V2 (tolvaptan, lixivaptan) selectivity or non-selective activity (conivaptan) which may be advantageous in some disorders. The V1a/V2 non-selective vasopressin antagonist conivaptan is the first vaptan which is approved by the FDA for the treatment of euvolaemic hyponatraemia. Received 3 February 2006; received after revision 16 March 2006; accepted 26 April 2006     

Comparison of some biological activities of arginine vasotocin and synthetic analogs

Zusammenfassung Nachweis, dass die Substitution in Positionen 3 und 8 im Arginin Vasotocin ([8-Arginin]-Oxytocin) selektive Veränderungen verschiedener biologischer Aktivitäten hervorruft, was am Modell der Oxytocinkonformation diskutiert wird.

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Supported in part by USPHS grant No. AM-13567.

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Neurohypophyseal hormone analogs are denoted in accordance with IUPAC-IUB Tentative Rules on Biochemical Nomenclature, Biochemistry6, 362 (1967) and J. biol. Chem.247, 977 (1972). All optically active amino acids are of thel-configuration unless otherwise noted. Arginine vasopressin, [8-arginine]vasopressin; lysine vasopressin, [8-lysine]vasopressin; AVT, arginine vasotocin, [8-arginine]oxytocin; Mpr, -mercaptopropionic acid.

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Acknowledgment. We thank Dr.Klaus Lübke, Schering AG, West Germany, for generously supplying us with the hormone analogs.     

The use of a Quantimet image analysis system to analyse trypsin banding patterns of V79/4 (AH1) Chinese hamster chromosomes

Summary A Quantimet image-analysis system was programmed to identify and print out banding patterns from G-banded chromosome spreads of a clone derived from the established cell line V79/4 (Chinese hamster fibroblasts), designated V79/4(AH1).Acknowledgments. We thank Dr. J.R.K. Savage and Dr J. Thacker, MRC Harwell for their advice, also Dr. P.D. Holt for useful discussions. We also thank D. Coates for his assistance with the Quantimet. This work was partly supported by Euratom grant No. 134-74-1 BIOUK.     

4-Proline and 4-hydroxyproline analogs of arginine vasopressin: role of the proline substitution in the two beta-turns of vasopressin

[4-L-Proline]arginine vasopressin, [4-D-proline]arginine vasopressin, [4-hydroxyproline]arginine vasopressin and [4-proline, 7-hydroxyproline] arginine vasopressin were synthesized and found to have antidiuretic activities of 91 +/- 4, 1.7 +/- 0.2, 1.0 +/- 0.1 and 4.4 +/- 1.0 units/mg, respectively. None of these analogs exhibited a significant level of rat pressor activity. The observed activities of these and other analogs with substitutions at position 4 and/or 7 are discussed on the basis of hypotheses and data bearing on the solution conformation of vasopressins.     

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.     

Annexin V interactions with collagen

Annexin V was originally identified as a collagen-binding protein called anchorin CII and was isolated from chondrocyte membranes by affinity chromatography on native type II collagen. The binding of annexin V to native collagen type II is stable at physiological ionic strength when annexin V is reconstituted in liposomes. The binding to native collagen types II and X, and to some extent to type I as well, was confirmed using recombinant annexin V. A physiological role for annexin V interactions with extracellular collagen is consistent with the localization of annexin V on the outer cell surface of chondrocytes, microvilli of hypertrophic chondrocytes, fibroblasts and osteoblasts. A breakthrough in our understanding of the function of annexin V was made with the discovery of its calcium channel activity. At least one of several putative functions of annexin V became obvious from studies on matrix vesicles derived from calcifying cartilage. It was found that calcium uptake by matrix vesicles depend on collagen type II and type X binding to annexin V in the vesicles and was lost when collagens were digested with collagenase; calcium influx was reconstituted after adding back native collagen II or V. These findings indicate that annexin V plays a major role in matrix vesicle-initiated cartilage calcification as a collagen-regulated calcium channel.     

Decreased levels of complex III core protein 1 and complex V beta chain in brains from patients with Alzheimer's disease and Down syndrome

Ubiquinol:cytochrome c oxidoreductase (complex III) and ATP synthase (complex V) are important enzymes in the mitochondrial electron transport chain. Defects in mitochondrial respiratory enzymes have been reported for several neurodegenerative diseases. In this study, we applied the proteomic approach to investigate protein levels of complex III core protein and complex V beta chain in brain regions of Alzheimer's disease (AD) and Down syndrome (DS) patients. Complex III core protein 1 was significantly reduced in the temporal cortex of AD patients. Complex V beta chain was significantly reduced in the frontal cortex of DS patients. We conclude that decreased mitochondrial respiratory enzymes could contribute to the impairment of energy metabolism observed in DS. These decreases could also cause the generation of reactive oxygen species and neuronal cell death (apoptosis) in DS as well as AD.     

Porcine factor V: cDNA cloning, gene mapping, three-dimensional protein modeling of membrane binding sites and comparative anatomy of domains

Factor V is a plasma protein essential for blood coagulation. This protein is involved in activated protein C resistance, the most common inherited thrombotic disorder known. We utilized the polymerase chain reaction to clone the porcine factor V gene by generating overlapping clones amplified with primers chosen by comparison with known nucleotide sequences. The porcine factor V cDNA contig encodes a predicted 2258-amino acid protein, making it the largest in comparison to the bovine, human, and murine proteins. Porcine factor V has the highest level of homology with bovine factor V, but also has high levels of conservation of important residues with all the species. Radiation hybrid mapping assigned the porcine factor V gene to chromosome 4. Three-dimensional models of factor V were generated and used to analyze membrane-binding sites in terms of conserved, and therefore likely important residues. Received 3 October 2000; revised 23 November 2000; accepted 6 December 2000     

Antihypertensive agents III. Synthesis and antihypertensive activity of 3-[4-(p-fluoro-phenyl)-1,2,3,6-tetrahydro-1-pyridyl]-1-[1-(2-hydroxyethyl)-5-methyl-4-pyrazolyl]-1-propanone

Zusammenfassung 3-[4-(p-Fluorphenyl)-1,2,3,6-te-trahydro-1-pyridyl]-1-[1-(2-hydroxyaethyl)-5-methyl-4-pyrazolyl]-1-propanon (I, CIBA 4416/B-Go) senkt bei normotonischen und hypertonischen Tieren den Blutdruck. Die Drucksenkung kann hauptsächlich auf die periphere Vasodilatation sowie auf die Vasomotorenzentren bezogen werden. Ausserdem wirkt CIBA 4416/B-Go adrenolytisch.

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Previous paper:V. P. Arya, R. S. Grewal, C. L. Kaul, S. P. Ghate, D. V. Mehta andT. George, J. Pharm. Sci.58, 432 (1969).Contribution No. 243 from CIBA Research Centre.     

High potassium intake increases the plasma concentration and urinary excretion of vasopressin in the rat

Summary The effect of alterations of dietary potassium intake on the plasma concentration and the urinary excretion of vasopressin was studied in male rats. Ingestion of a high potassium diet resulted in increases in the plasma concentrations of potassium and vasopressin, systolic blood pressure, urine flow, and urinary vasopressin excretion. Ingestion of a low potassium diet had little effect on the plasma vasopressin concentration and systolic blood pressure but caused decreases in the plasma potassium concentration and urinary vasopressin excretion. The results indicate that physiological changes in the plasma potassium concentration or some other consequence of altered dietary potassium intake can affect vasopressin release and excretion.     

High potassium intake increases the plasma concentration and urinary excretion of vasopressin in the rat

The effect of alterations of dietary potassium intake on the plasma concentration and the urinary excretion of vasopressin was studied in male rats. Ingestion of a high potassium diet resulted in increases in the plasma concentrations of potassium and vasopressin, systolic blood pressure, urine flow, and urinary vasopressin excretion. Ingestion of a low potassium diet had little effect on the plasma vasopressin concentration and systolic blood pressure but caused decreases in the plasma potassium concentration and urinary vasopressin excretion. The results indicate that physiological changes in the plasma potassium concentration or some other consequence of altered dietary potassium intake can affect vasopressin release and excretion.     

Coordinated shift of olfactory amino acid responses and V2R expression to an amphibian water nose during metamorphosis

All olfactory receptors identified in teleost fish are expressed in a single sensory surface, whereas mammalian olfactory receptor gene families segregate into different olfactory organs, chief among them the main olfactory epithelium expressing ORs and TAARs, and the vomeronasal organ expressing V1Rs and V2Rs. A transitional stage is embodied by amphibians, with their vomeronasal organ expressing more ‘modern’, later diverging V2Rs, whereas more ‘ancient’, earlier diverging V2Rs are expressed in the main olfactory epithelium. During metamorphosis, the main olfactory epithelium of Xenopus tadpoles transforms into an air-filled cavity (principal cavity, air nose), whereas a newly formed cavity (middle cavity) takes over the function of a water nose. We report here that larval expression of ancient V2Rs is gradually lost from the main olfactory epithelium as it transforms into the air nose. Concomitantly, ancient v2r gene expression begins to appear in the basal layers of the newly forming water nose. We observe the same transition for responses to amino acid odorants, consistent with the hypothesis that amino acid responses may be mediated by V2R receptors.     

Long term stability of lysine vasopressin and of specifically tritiated lysine vasopressin in weakly acidic aqueous solutions

Summary Lysine vasopressin tritiated at meta position of tyrosine (1.9 Ci/mmole) decomposes during a long storage period, mainly as a result of radiolysis. The loss of specific activity by isotopic exchange with water is slow. The nonlabelled peptide undergoes only very minute decomposition (polymerization). Specific radioactivity declines with a half life of 47.4 months.Acknowledgments. This research was supported by Swiss National Science Foundation, grant No.3.040.76 (V.P.). Some of the measurements (years 1969–70) were carried out at Roche Institute of Molecular Biology, Nutley, N.J., USA.     

Endonuclease V: an unusual enzyme for repair of DNA deamination

Endonuclease V (endo V) was first discovered as the fifth endonuclease in Escherichia coli in 1977 and later rediscovered as a deoxyinosine 3′ endonuclease. Decades of biochemical and genetic investigations have accumulated rich information on its role as a DNA repair enzyme for the removal of deaminated bases. Structural and biochemical analyses have offered invaluable insights on its recognition capacity, catalytic mechanism, and multitude of enzymatic activities. The roles of endo V in genome maintenance have been validated in both prokaryotic and eukaryotic organisms. The ubiquitous nature of endo V in the three domains of life: Bacteria, Archaea, and Eukaryotes, indicates its existence in the early evolutionary stage of cellular life. The application of endo V in mutation detection and DNA manipulation underscores its value beyond cellular DNA repair. This review is intended to provide a comprehensive account of the historic aspects, biochemical, structural biological, genetic and biotechnological studies of this unusual DNA repair enzyme.     

Myosin V from head to tail

Myosin V (myoV), a processive cargo transporter, has arguably been the most well-studied unconventional myosin of the past decade. Considerable structural information is available for the motor domain, the IQ motifs with bound calmodulin or light chains, and the cargo-binding globular tail, all of which have been crystallized. The repertoire of adapter proteins that link myoV to a particular cargo is becoming better understood, enabling cellular transport processes to be dissected. MyoV is processive, meaning that it takes many steps on actin filaments without dissociating. Its extended lever arm results in long 36-nm steps, making it ideal for single molecule studies of processive movement. In addition, electron microscopy revealed the structure of the inactive, folded conformation of myoV when it is not transporting cargo. This review provides a background on myoV, and highlights recent discoveries that show why myoV will continue to be an active focus of investigation. Received 31 October 2007; received after revision 4 December 2007; accepted 2 January 2008     

Folding and assembly defects of pyruvate dehydrogenase deficiency-related variants in the E1α subunit of the pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.     

High and low affinity transport ofL-arginine in rat brain synaptosomes

The uptake ofL-arginine into purified rat brain synaptosomes was investigated with respect to time and various concentrations ofL-[³H] arginine. Specific uptake was found to be linear with time for up to 5 min of incubation at 37°C. Electrolytes, including sodium chloride, potassium chloride, magnesium chloride and calcium chloride, inhibited uptake of 3 ML-arginine, and the inhibitory effect increased with increased electrolyte concentration under constant osmolarity. It was found thatL-arginine was transported into synaptosomes by two uptake components — a high affinity component (3.5 M) and a low affinity component (100 M). These two components were similar to the Ly⁺ system because of their extreme sensitivity to inhibition byL-lysine andL-ornithine but were distinguishable from each other by kinetic analysis of the uptake data and by their relative sensitivity to inhibition by several amino acids.     

Heilmittelchemische Studien mit Dien-Additionsprodukten V: Über die coronardilatierenden Eigenschaften einer neuen Klasse von Aminen

Summary The addition productsof 2 M N-alkyl substituted maleinimides with 1M asymmetric diphenylethylen and some of its substitution products were reduced with LiAlH₄ to give amines of the structure V. These substances produce coronary dilation on the isolated guinea-pig heart perfused according to the method of Langendorff, a few of them being more active than papaverin. On the barbiturate anaesthetized dog, however, the activity was much less than that of papaverin after intraarterial injection and was entirely absent on intravenous application. High doses caused lowering of the blood pressure. The limited value of the Langendorff heart preparation for screening coronary dilators is emphasized.

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IV. Mitteilung: I.Molnar, Helv. Chim Acta49, 586 (1966).