Alpha-LNA (alpha-D-configured locked nucleic acid)

Two pyrimidine alpha-LNA nucleoside monomers have been synthesised and incorporated into alpha-configured oligonucleotides. A fully modified mixed alpha-LNA sequence displays unprecedented parallel stranded hybridisation with complementary RNA and a remarkable selectivity for RNA over DNA. Modelling shows alpha-LNA:RNA to form an extended duplex with a very broad major groove.     

Bi- and tricyclic nucleoside derivatives restricted in S-type conformations and obtained by RCM-reactions

Ring-closing metathesis (RCM) is applied as a new and powerful technology in the construction of nucleoside analogues that are conformationally restricted in S-type conformations due to additional 3',4'- and/or 3',5'-linkages.     

Autosomal dominant familial neurohypophyseal diabetes insipidus

Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disorder caused by progressive postnatal arginine vasopressin (AVP) deficiency resulting from mutations in the AVP gene encoding the AVP pre-prohormone. It has been suggested that these mutations exert their effect on the cellular handling of the AVP prohormone by leading to the synthesis of mutant hormone precursor that fails to be processed and/or folded properly in the endoplasmic reticulum (ER). As a consequence, it is retained by the ER protein quality control machinery resulting in protein accumulation and initiation of cellular processes leading to degeneration of the AVP producing neuron. This review summarizes the current knowledge on adFNDI and discusses different hypotheses concerning its pathogenesis.     

Endurance training and GH administration in elderly women: effects on abdominal adipose tissue lipolysis

In the present study, the effect of endurance training alone and endurance training combined with recombinant human growth hormone (rhGH) administration on subcutaneous abdominal adipose tissue lipolysis was investigated. Sixteen healthy women [age 75 +/- 2 yr (mean +/- SE)] underwent a 12-wk endurance training program on a cycle ergometer. rhGH was administered in a randomized, double-blinded, placebo-controlled design in addition to the training program. Subcutaneous abdominal adipose tissue lipolysis was estimated by means of microdialysis combined with measurements of subcutaneous abdominal adipose tissue blood flow (ATBF; (133)Xe washout). Whole body fat oxidation was estimated simultaneously by indirect calorimetry. Before and after completion of the training program, measurements were performed both at rest and during 60 min of continuous cycling at a workload corresponding to 60% of pretraining peak oxygen uptake. Endurance training alone did not affect subcutaneous abdominal adipose tissue lipolysis either at rest or during exercise, as reflected by identical levels of interstitial adipose tissue glycerol, subcutaneous abdominal ATBF, and plasma nonesterified fatty acids before and after completion of the training program. Similarly, no effect on subcutaneous abdominal adipose tissue lipolysis was observed when combining endurance training with rhGH administration. However, in both the placebo and the GH groups, fat oxidation was significantly increased during exercise performed at the same absolute workload after completion of the training program. We conclude that the changed lipid metabolism during exercise observed after endurance training alone or after endurance training combined with rhGH administration is not due to alterations in subcutaneous abdominal adipose tissue metabolism in elderly women.     

Calculation of electric fields induced in the human knee by a coil applicator

Calculations are presented of the induced electric fields and current densities in the cartilage of the knee produced by a coil applicator developed for applying pulsed magnetic fields to osteoarthritic knees. This applicator produces a sawtooth-like magnetic field waveform composed of a series of 260-micros pulses with a peak to peak magnitude of approximately 0.12 mT in the cartilage region. The simulations were performed using a recently developed 3 dimensional finite difference frequency domain technique for solving Maxwell's equations with an equivalent circuit model. The tissue model was obtained from the anatomically segmented human body model of Gandhi. The temporal peak electric field magnitude was found to be -153 mV/m, averaged within the medial cartilage of the knee for the typical dB/dt excitation levels of this coil. The technique can be extended to analyze other excitation waveforms and applicator designs.     

Central volume expansion is pivotal for sustained decrease in heart rate during seated to supine posture change

During prolonged, static carotid baroreceptor stimulation by neck suction (NS) in seated humans, heart rate (HR) decreases acutely and thereafter gradually increases. This increase has been explained by carotid baroreceptor adaptation and/or buffering by aortic reflexes. During a posture change from seated to supine (Sup) with similar carotid stimulation, however, the decrease in HR is sustained. To investigate whether this discrepancy is caused by changes in central blood volume, we compared (n = 10 subjects) the effects of 10 min of seated NS (adjusted to simulate carotid stimulation of a posture change), a posture change from seated to Sup, and the same posture change with left atrial (LA) diameter maintained unchanged by lower body negative pressure (Sup + LBNP). During Sup, the prompt decreases in HR and mean arterial pressure (MAP) were sustained. HR decreased similarly within 30 s of NS (65 +/- 2 to 59 +/- 2 beats/min) and Sup + LBNP (65 +/- 2 to 58 +/- 2 beats/min) and thereafter gradually increased to values of seated. MAP decreased similarly within 5 min during Sup + LBNP and NS (by 7 +/- 1 to 9 +/- 1 mmHg) and thereafter tended to increase toward values of seated subjects. Arterial pulse pressure was increased the most by Sup, less so by Sup + LBNP, and was unchanged by NS. LA diameter was only increased by Sup. In conclusion, static carotid baroreceptor stimulation per se causes the acute (<30 s) decrease in HR during a posture change from seated to Sup, whereas the central volume expansion (increased LA diameter and/or arterial pulse pressure) is pivotal to sustain this decrease. Thus the effects of central volume expansion override adaptation of the carotid baroreceptors and/or buffering of aortic reflexes.     

Atrial distension, arterial pulsation, and vasopressin release during negative pressure breathing in humans

During an antiorthostatic posture change, left atrial (LA) diameter and arterial pulse pressure (PP) increase, and plasma arginine vasopressin (AVP) is suppressed. By comparing the effects of a 15-min posture change from seated to supine with those of 15-min seated negative pressure breathing in eight healthy males, we tested the hypothesis that with similar increases in LA diameter, suppression of AVP release is dependent on the degree of increase in PP. LA diameter increased similarly during the posture change and negative pressure breathing (-9 to -24 mmHg) from between 30 and 31 +/- 1 to 34 +/- 1 mm (P < 0.05). The increase in PP from 38 +/- 2 to 44 +/- 2 mmHg (P < 0.05) was sustained during the posture change but only increased during the initial 5 min of negative pressure breathing from 36 +/- 3 to 42 +/- 3 mmHg (P < 0.05). Aortic transmural pressure decreased during the posture change and increased during negative pressure breathing. Plasma AVP was suppressed to a lower value during the posture change (from 1.5 +/- 0.3 to 1.2 +/- 0.2 pg/ml, P < 0.05) than during negative pressure breathing (from 1.5 +/- 0.3 to 1.4 +/- 0.3 pg/ml). Plasma norepinephrine was decreased similarly during the posture change and negative pressure breathing compared with seated control. In conclusion, the results are in compliance with the hypothesis that during maneuvers with similar cardiac distension, suppression of AVP release is dependent on the increase in PP and, furthermore, probably unaffected by static aortic baroreceptor stimulation.     

Effect of oxytocin receptor blockade on rat myometrial responsiveness to prostaglandin f(2)(alpha)

In the present study we have shown that the genetic expression of prostaglandin (PG)F(2alpha) receptor (R) and cyclooxygenase (COX)-2 increases in laboring rat myometrium. This finding was associated with a relatively weak contractile in vitro response (E:(max)) of isolated uterine strips when challenged with PGF(2alpha). Five days postpartum PGF(2alpha)-R mRNA values exceeded those during labor while COX-2 mRNA was reduced to preparturient values. Maximal contractility of isolated strips stimulated with PGF(2alpha) at this time was enhanced and E:C(50) decreased. Oxytocin treatment of estrogen-primed nonpregnant rats down-regulated uterine contractile responsiveness to PGF(2alpha), leaving mRNA values for this receptor unchanged, whereas oxytocin receptor blockade with atosiban (an oxytocin receptor antagonist) left E:(max) unaltered. In contrast, atosiban treatment of pregnant rats resulted in a 2.5-fold increase in E:(max) and a considerably reduced EC(50) during labor when compared to untreated delivering rats. The increased contractile ability was associated with a threefold increase in PGF(2alpha)-R mRNA production, indicating that the regulation by atosiban of the PGF(2alpha)-induced response is exerted at the genetic level. Based on the present data we suggest that 1) PGF(2alpha)-R stimulation may not primarily exert a contracting role in the normally delivering myometrium, and 2) the presence of the PGF(2alpha)-R system in rat myometrium may explain the apparent functional redundancy of the oxytocinergic system during the process of birth in animals lacking oxytocin or where the oxytocin receptor is blocked. In this context PGF(2alpha) receptor stimulation may, in the absence of oxytocin receptor stimulation, exert the contractile forces needed for proper propulsion of the fetus.     

Specific binding of alpha-macroglobulin to complement-type repeat CR4 of the low-density lipoprotein receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large surface receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites are located in clusters of complement-type repeats (CR), where the general absence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to the LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglobulin (alpha(2)M). To determine the major binding epitope(s) in the LRP, we generated a complete set of tandem CR proteins spanning the second cluster of CR domains, and identified a binding site for alpha(2)M in the N-terminal part of the cluster comprising CR3-CR6, using ligand blotting and surface plasmon resonance (SPR) analysis. The specific site involved in alpha(2)M recognition resides in the fourth CR domain, CR4, whereas another site is identified in CR5. An acidic epitope in CR4 is identified as important for binding alpha(2)M by mutagenesis and SPR analysis. The formation of the complex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is characterized by analytical size-exclusion chromatography, which demonstrates a sufficiently strong interaction between the alpha(1)M RBD and CR34 or CR45 for the isolation of a complex.     

8-Substituted cAMP analogues reveal marked differences in adaptability, hydrogen bonding, and charge accommodation between homologous binding sites (AI/AII and BI/BII) in cAMP kinase I and II

cAMP analogues, systematically substituted at position 8 of the adenine moiety (C8), were tested quantitatively for binding to each cAMP interaction site (A and B) of the regulatory subunits of cAMP-dependent protein kinase type I (RI) and II (RII). Site AII did not accommodate cAMP analogues with any bulk at position 8, whereas site AI accepted even bulky 8-substituents. This implies that the narrow, buried pocket of site AI facing position C8 of cAMP in the RI-cAMP crystal [Su, Y., Dostmann, W. R., Herberg, F. W., Durick, K., Xuong, N. H., Ten Eyck, L., Taylor, S. S., and Varughese, K. I. (1995) Science 269, 807-813] must undergo considerable conformational change and still support high-affinity cAMP analogue binding. The B sites of RI and RII differed in three respects. First, site BI had a lower affinity than site BII for cAMP analogues with hydrophobic, bulky 8-substituents. Second, site BI had a preference for substituents with hydrogen bonding donor potential close to C8, whereas site BII had a preference for substituents with hydrogen bonding acceptor potential. This implies that Tyr(371) of RI and the homologous Tyr(379) of RII differ in their hydrogen bonding preference. Third, site BI preferred analogues with a positively charged amino group that was an extended distance from C8, whereas site BII discriminated against a positive charge. The combined results allow refinement of the cAMP binding site geometry of RI and RII in solution, and suggest design of improved isozyme-specific cAMP analogues.