A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Combining optical and electrical impedance techniques for quantitative measurement of confluence in MDCK-I cell cultures

A new method combining optical and electrical impedance measurements is described that enables submicroscopic cell movements to be monitored. The cells are grown on small gold electrodes that are transparent to light. This modified electrical cell-substrate impedance sensor (ECIS) allows simultaneous microscopic recording of both growth and motility, thus enabling cell confluence on the electrodes to be systematically correlated to the impedance in regular time intervals of seconds and for extended periods of time. Furthermore, the technique provides an independent measure of monolayer cell densities that we compare to calculated values from a theoretical model. We have followed the attachment and spreading behavior of epithelial Madin-Darby canine kidney strain I (MDCK-I) cell cultures on microelectrodes for up to 40 h. The studies reveal a high degree of correlation between the measured resistance at 4 kHz and the corresponding cell confluence in 4- to 6-h intervals with typical linear cross-correlation factors of r equaling approximately 0.9. In summary, the impedance measured with the ECIS technique provides a good quantitative measure of cell confluence.     

Beta-alanine containing peptides: a novel molecular tool for the design of gamma-turns.

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution conformational characterization of the cyclic tetrapeptide cyclo-(L-Pro-beta-Ala-L-Pro-beta-Ala). This peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide (DCCI). The compound crystallizes in the orthorombic space group P2(1)2(1)2(1) from ethyl acetate. All peptide bonds are trans. The molecular conformation is stabilized by two intramolecular hydrogen bonds between the CO and NH groups of the two beta-alanine residues. These hydrogen bonds take place in a C7 structure in which both proline residues occupy the 2 position of an inverse gamma-turn. The two beta-alanine residues have a typical folded conformation (around the C alpha-C beta bond) observed in other cyclic peptides containing this residue. A detailed 1H-nmr analysis in CD3CN solution has been carried out. The molecule assumes a twofold symmetry in solution with a molecular conformation consistent with that observed in the solid state.     

An Overview of the Genetic Structure within the Italian Population from Genome-Wide Data

In spite of the common belief of Europe as reasonably homogeneous at genetic level, advances in high-throughput genotyping technology have resolved several gradients which define different geographical areas with good precision. When Northern and Southern European groups were considered separately, there were clear genetic distinctions. Intra-country genetic differences were also evident, especially in Finland and, to a lesser extent, within other European populations. Here, we present the first analysis using the 125,799 genome-wide Single Nucleotide Polymorphisms (SNPs) data of 1,014 Italians with wide geographical coverage. We showed by using Principal Component analysis and model-based individual ancestry analysis, that the current population of Sardinia can be clearly differentiated genetically from mainland Italy and Sicily, and that a certain degree of genetic differentiation is detectable within the current Italian peninsula population. Pair-wise F_ST statistics Northern and Southern Italy amounts approximately to 0.001 between, and around 0.002 between Northern Italy and Utah residents with Northern and Western European ancestry (CEU). The Italian population also revealed a fine genetic substructure underscoring by the genomic inflation (Sardinia vs. Northern Italy = 3.040 and Northern Italy vs. CEU = 1.427), warning against confounding effects of hidden relatedness and population substructure in association studies.     

Solid state structural analysis of the cyclooctapeptide cyclo- (Pro1-Pro-Phe-Phe-Ac6c-Ile-D-Ala-Val8)

A solid state analysis of the cyclic octapeptide c(-Pro(1)-Pro-Phe-Phe-Ac(6)c-Ile-D-Ala-Val(8)-) (C8-CLA), containing the Pro-Pro-Phe-Phe sequence, followed by the bulky helicogenic C(alpha,alpha)-dialkylated 1-aminocyclohexane-1-carboxylic acid (Ac(6)c) residue and a D-Ala residue in position 7, has been carried out by x-ray diffraction.The crystals, grown from a DMSO solution, are monoclinic, space group P2(1) with a = 13.458(3) A, b = 19. 404(5) A, c = 21.508(4) A, and beta = 90.83(6) degrees, with two independent cyclic molecules in the asymmetric unit, two DMSO molecules, and three water molecules. The structure has been solved using the half and bake procedure by Sheldrick, and refined to final R1 and wR2 indices of 0.0613 and 0.1534 for 9867 reflections with I > 2sigma(I).This cyclic peptide, a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A [c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), CLA] has been designed to study the influence of the ring size reduction on the conformational behavior of CLA and more in general to obtain structural information on asymmetric cyclic octapeptides.The compound exhibits, in the solid state, a "banana-twisted" conformation with a cis peptide bond located between the two proline residues. Five intramolecular H bonds stabilize the structure: one type VIa beta-turn, two consecutive type III/I beta-turns, one gamma-turn, and one C(16) bend.The structure has also been compared with either the solution structure previously reported by us and obtained by nmr and computational analysis, and with solid state structural data reported in the literature on cyclic octapeptides.     

Apoptosis meets proteasome, an invaluable therapeutic target of anticancer drugs

This report reviews the current status of extensive efforts directed towards the interpretation of crosstalk between apoptosis and proteasome to understanding the molecular mechanism of anticancer agents targeting proteasome, with particular focus on MG132 and PS-341. The discovery that all cancer cells have retained the apoptotic death program has offered to the researchers new biochemical targets to design anticancer drugs. Moreover, the demonstration that proteasome inhibition induces apoptosis and sensitizes cancer cells to traditional tumoricidal agents has proposed the proteasome as an attractive target for development of new anticancer drugs. Since then, a number of both naturally occurring and synthetic inhibitors of the proteasome have been identified. The best characterized and most widely used inhibitors of the proteasome are the peptide aldehydes; among these MG132, due to its broad spectrum of action, low cost and rapid reversibility of action, still remains the first choice to study proteasome function in cell and tissue cultures. Recently, a very potent new class of selective and reversible proteasome inhibitors which contains an inhibitory boronate group has been described. PS-341 represent the first of this promising class of agents that could have application in cancer therapy and it is the only that has progressed to clinical trials.     

Postnatal ontogeny of glucose homeostasis and insulin action in sheep

Glucose tolerance declines with maturation and aging in several species, but the time of onset and extent of changes in insulin sensitivity and insulin secretion and their contribution to changes in glucose tolerance are unclear. We therefore determined the effect of maturation on glucose tolerance, insulin secretion, and insulin sensitivity in a longitudinal study of male and female sheep from preweaning to adulthood, and whether these measures were related across age. Glucose tolerance was assessed by intravenous glucose tolerance test (IVGTT, 0.25 g glucose/kg), insulin secretion as the integrated insulin concentration during IVGTT, and insulin sensitivity by hyperinsulinemic-euglycemic clamp (2 mU insulin.kg(-1).min(-1)). Glucose tolerance, relative insulin secretion, and insulin sensitivity each decreased with age (P < 0.001). The disposition index, the product of insulin sensitivity, and various measures of insulin secretion during fasting or IVGTT also decreased with age (P < 0.001). Glucose tolerance in young adult sheep was independently predicted by insulin sensitivity (P = 0.012) and by insulin secretion relative to integrated glucose during IVGTT (P = 0.005). Relative insulin secretion before weaning was correlated positively with that in the adult (P = 0.023), whereas glucose tolerance, insulin sensitivity, and disposition indexes in the adult did not correlate with those at earlier ages. We conclude that glucose tolerance declines between the first month of life and early adulthood in the sheep, reflecting decreasing insulin sensitivity and absence of compensatory insulin secretion. Nevertheless, the capacity for insulin secretion in the adult reflects that early in life, suggesting that it is determined genetically or by persistent influences of the perinatal environment.