Structural and electrophysiological effects of local anesthetics and of low temperature on myelinated nerves: implication of the lipid chains in nerve excitability

X-ray scattering and electrophysiological experiments performed on toad sciatic nerves as a function of the exposure to either low temperature or tetracaine yielded the following results: (i) the main structural effect is to thicken the individual membranes, thus to stiffen the acyl chains and increase the repeat distance of the one-dimensional lattice, phenomena that are typical of lipid-containing systems with disordered chains; (ii) the electrophysiological effect is to decrease the amplitude and velocity of the compound action potential; (iii) the structural and physiological effects of the two agents are practically identical. Since the structural and the electrophysiological parameters have different origins in the nerves (the structure regards the myelin sheath, the electrical signals originate at the nodes of Ranvier) it is inferred that tetracaine and low temperature exert similar effects on the membranes of both the myelin sheath and the nodes of Ranvier. Also, since local anesthetics act by inhibiting the Na+ channels, these observations suggest that the acyl chain conformation modulates the channel function and thus the generation of action potential.     

The assembly of beta-methylene-TAD, a metabolically stable analogue of the antitumor agent TAD, by the stepwise esterification of monodeprotected methylenebis-(phosphonate) benzyl esters under Mitsunobu conditions

Synthesis of the metabolically stable analogue of thiazole-4-carboxamide adenine dinucleotide (beta-methylene-TAD) was achieved via the sequential monodeprotection of tetrabenzyl methylenebis(phosphonate) after two rounds of Mitsunobu esterifications with the corresponding nucleoside components, tiazofurin and adenosine.     

Different signaling pathways in bovine sperm regulate capacitation and hyperactivation

Hyperactivated sperm motility is characterized by high-amplitude and asymmetrical flagellar beating that assists sperm in penetrating the oocyte zona pellucida. Other functional changes in sperm, such as activation of motility and capacitation, involve cross talk between the cAMP/PKA and tyrosine kinase/phosphatase signaling pathways. Our objective was to determine the role of the cAMP/protein kinase A (PKA) signaling pathway in hyperactivation. Western blot analyses of detergent extracts of whole sperm and flagella were performed using antiphosphotyrosine antibody. Bull sperm capacitated by 10 microg/ml heparin and/or 1 mM dibutyryl-cAMP plus 100 microM 3-isobutyl-1-methylxanthine exhibited increased protein tyrosine phosphorylation without becoming hyperactivated. Procaine (5 mM) or caffeine (10 mM) immediately induced hyperactivation in nearly 100% of motile sperm but did not increase protein tyrosine phosphorylation. After 4 h of incubation with caffeine, sperm expressed capacitation-associated protein tyrosine phosphorylation but hyperactivation was significantly reduced. Sperm initially hyperactivated by procaine or caffeine remained hyperactivated for at least 4 h in the presence of Rp-cAMPS (cAMP antagonist) or PKA inhibitors H-89 or H-8. Pretreatment with inhibitors also failed to block induction of hyperactivation; however, the inhibitors did block protein tyrosine phosphorylation when sperm were incubated with capacitating agents, thereby verifying inhibition of the cAMP/PKA pathway. While induction of hyperactivation did not depend on cAMP/PKA, it did require extracellular Ca(2+). These findings indicate that hyperactivation is mediated by a Ca(2+) signaling pathway that is separate or divergent from the pathway associated with acquisition of acrosomal responsiveness and does not involve protein tyrosine phosphorylation downstream of the actions of procaine or caffeine.     

Synthesis, stability, and protonation studies of a self-complementary dodecamer containing the modified nucleoside 2'-deoxyzebularine

The nucleoside 2'-deoxyzebularine (K) was incorporated into the self-complementary dodecamer 5'-CGTACGKGTACG-3' by solid-phase 2-cyanoethylphosphoramidite chemistry using dimethoxytrityl (DMT) as the 5'-hydroxyl protecting group. Standard synthesis cycles using trichloroacetic acid and short ammonia treatment (50 degrees C for 30 min) were found to be the optimal conditions to obtain the desired dodecamer with minimum acid and basic degradation of the acid- and base-sensitive 2-pyrimidinone residue. The protonation equilibria of the K nucleoside and of the dodecamer at 37 degrees C were studied by means of spectroscopically monitored titrations. For the K nucleoside, a pK(a) value of 3.13 +/- 0.09 was obtained. For the dodecamer, four acid-base species were found in the pH range 2-12, with pK(a) values of 9.60 +/- 0.07, 4.46 +/- 0.16, and 2.87 +/- 0.19. Melting experiments were carried out to confirm the proposed acid-base concentration profiles. Finally, kinetic experiments were also carried out at several pH values to evaluate the stability of the K nucleoside and of the dodecamer. An increased stability was shown by the K nucleoside when incorporated into the dodecamer. Multivariate methods based on both hard- and soft-modeling were applied for the analysis of spectroscopic data, allowing the estimation of concentration profiles and pure spectra.     

Development of isothiocyanate-enriched broccoli,and its enhanced ability to induce phase 2 detoxification enzymes in mammalian cells

Broccoli florets contain low levels of 3-methylsuphinylpropyl and 4-methylsulphinylbutyl glucosinolates. Following tissue disruption, these glucosinolates are hydrolysed to the corresponding isothiocyanates (ITCs), which have been associated with anticarcinogenic activity through a number of physiological mechanisms including the induction of phase II detoxification enzymes and apoptosis. In this paper, we describe the development of ITC-enriched broccoli through the introgression of three small segments of the genome of Brassica villosa, a wild relative of broccoli, each containing a quantitative trait locus (QTL), into a broccoli genetic background, via marker-assisted selection and analysis of glucosinolates in the florets of backcross populations. Epistatic and heterotic effects of these QTLs are described. The ITC-enriched broccoli had 80-times the ability to induce quinone reductase (a standard assay of phase II induction potential) when compared to standard commercial broccoli, due both to an increase in the precursor glucosinolates and a greater conversion of these into ITCs.     

Synthesis and conformational analysis of a locked analogue of carbovir built on a bicyclo[3.1.0]hex-2-enyl template

The synthesis and biological evaluation of a carbovir analogue (5) built on a bicyclo[3.1.0]hex-2-enyl template is described. A conformational analysis using density functional theory at the B3LYP/6-31G* level has been carried out on the rigid pseudosugar template of 5, the cyclopentene moiety of carbovir and the bicyclo[3.1.0]hex-2-yl pseudosugars of two isomeric carbonucleosides (12 and 13) containing exo- and endo-fused cyclopropane rings. The results show that while the planar configuration of the fused cyclopentane ring of compound 5 helps retain weak anti-HIV activity, the ability of the cyclopentene ring of carbovir to easily adopt a planar or puckered conformation with little energy penalty may prove to be a crucial advantage. The bicyclo[3.1.0]hex-2-yl nucleosides 12 and 13 that were inactive against HIV exhibited stiffer resistance to having a planar, fused cyclopentane moiety.     

Laser Raman spectra of glucagon

Laser Raman spectroscopy study indicates that in concentrated fresh acidic solution (30 mg/ml), glucagon remains predominantly α-helix and not random-coil. The splitting of the amide III band into three components in the crystal at 1262, 1275, and 1295 cm^?1 is due to the α-conformation as expected. The presence of a small fraction of β-conformation is demonstrated by the appearance of the weak band at 1230 cm^?1 in the fresh solution. This study also established the frequencies of amide III′ bands for the α- and β-conformations of glucagon: 957 and 988 cm^?1 for α and β forms, respectively. The conformations of acidic and basic glucagon solutions are apparently different.