Initial orthostatic hypotension in teenagers and young adults.

OBJECTIVE: To assess: (1) the frequency of an abnormally large fall in blood pressure (BP) upon standing from supine in patients with initial orthostatic hypotension (IOH); (2) the underlying hemodynamic mechanisms of this fall in BP upon standing from supine and from squatting. METHODS: In a retrospective study of 371 patients (≤30 years) visiting the syncope unit, the hemodynamic response to standing and squatting were studied in 26 patients who were diagnosed clinically with IOH, based on history taking only. In six patients changes in cardiac output (CO) and systemic vascular resistance (SVR) were determined, and the underlying hemodynamics were analyzed. RESULTS: 15/26 (58 %) patients with IOH had an abnormally large initial fall in systolic BP (≥40 mmHg). There was a large scatter in CO and SVR response after arising from supine [ΔCO at BP nadir median -8 % (range -37, +27 %); ΔSVR at BP nadir median -31 % (range -46, +10 %)]. The hemodynamic response after squatting showed a more consistent pattern, with a fall in SVR in all six patients [ΔCO at BP nadir median +23 % (range -12, +31 %); ΔSVR at BP nadir median -42 %, (range -52, -35 %)]. INTERPRETATION: The clinical diagnosis of IOH is based on history taking, as an abnormally large fall in systolic BP can only be documented in 58 %. For IOH upon standing after supine rest, the hemodynamic mechanism can be either a large fall in CO or in SVR. For IOH upon arising from squatting a large fall in SVR is a consistent finding.

MSNA during prolonged post-faint hypotension.

BACKGROUND: Following tilt-induced syncope, blood pressure usually recovers rapidly after tilt back to the horizontal position. However, in some patients, hemodynamic recovery is delayed, a condition recently termed "prolonged post-faint hypotension" (PPFH). The mechanism is thought to be mediated by increased vagal outflow rather than exaggerated peripheral vasodilatation and sympathetic withdrawal. To date, no muscle sympathetic nerve activity (MSNA) recordings have been reported in this condition, so we aimed to confirm that neither vasodilatation nor MSNA withdrawal was responsible. OBJECTIVES: To retrospectively select patients with satisfactory recordings of continuous BP and MSNA during tilt-induced syncope. To compare hemodynamic and MSNA profiles in patients with PPFH to patients with normal recovery (NR) after tilt-back. METHODS: All patients were studied in Christchurch, New Zealand, between 1998 and 2008 using continuous arterial BP monitoring, and microneurographic recordings of MSNA from the right leg. Only patients with satisfactory BP and MSNA data throughout baseline, head-up tilt and presyncope were selected. Stroke volume (SV), cardiac output (CO), and total peripheral resistance (TPR) were derived using Modelflow. After baseline measurements, patients were tilted to the head-up 60° position and given GTN spray if asymptomatic after 20 min. Following the onset of presyncope, patients were tilted slowly back to the horizontal. PPFH was defined as systolic BP <85 mmHg for at least 2 min after tilt-back. Measurements were averaged at baseline, early tilt, presyncope, early and late recovery. Within-group comparisons were made between baseline and all other time points. Between-group comparisons were made over all time points. RESULTS: Patients with PPFH (7 males, age 46 ± 5 years, n = 8) and with NR (8 males, age 47 ± 6 years, n = 8) were selected. Presyncope was provoked by GTN in 4/8 patients in each group. In both groups, MAP remained below baseline during early and late recovery: PPFH 84 ± 5 versus 51 ± 5 and 64 ± 5 mmHg (p = 0.001, p = 0.001); NR 104 ± 5 versus 83 ± 5 and 93 ± 5 mmHg (p = 0.001, p = 0.03). However, MAP and HR were lower in the PPFH group (p = 0.004, p = 0.023). During early recovery, CO remained below baseline only in the PPFH group (p = 0.001), whereas TPR remained constant in both groups. In both groups, all MSNA indices tended to remain above baseline levels during early and late recovery. PPFH 25 ± 2 increased to 31 ± 6 and 29 ± 4 bursts/min (p = 0.09, 0.02); NR 23 ± 3 increased to 33 ± 3 and 34 ± 3 bursts/min (p = 0.06, 0.01). CONCLUSIONS: PPFH does not appear to be mediated by exaggerated vasodilatation or sympathetic withdrawal. Delayed recovery of cardiac output by increased vagal outflow is a more likely mechanism.

Prolonged post-faint hypotension can be reversed by dynamic tension.

A severe variant of vasovagal syncope, observed during tilt tests and blood donation has recently been termed "prolonged post-faint hypotension" (PPFH). A 49-year-old male with a life-long history of severe fainting attacks underwent head-up tilt for 20 min, and developed syncope 2 min after nitroglycerine spray. He was unconscious for 40 s and asystolic for 22 s. For the first 2 min of recovery, BP and HR remained low (65/45 mmHg and 40 beats/min) despite passive leg-raising. Blood pressure (and symptoms) only improved following active bilateral leg flexion and extension ("dynamic tension"). During PPFH, when vagal activity is extreme, patients may require central stimulation as well as correction of venous return.

Exceptional cryptic diversity and multiple origins of parthenogenesis in a freshwater ostracod.

The persistence of asexual reproduction in many taxa depends on a balance between the origin of new asexual lineages and the extinction of old ones. This turnover determines the diversity of extant asexual populations and so influences the interaction between sexual and asexual modes of reproduction. Species with mixed reproduction, like the freshwater ostracod (Crustacea) morphospecies Eucypris virens, are a good model to examine these dynamics. This species is also a geographic parthenogen, in which sexual females and males co-exist with asexual females in the circum-Mediterranean area only, whereas asexual females occur all over Europe. A molecular phylogeny of E. virens based on the mitochondrial COI and 16S fragments is presented. It is characterised by many distinct clusters of haplotypes which are either exclusively sexual or asexual, with only one exception, and are often separated by deep branches. Analysis of the phylogeny reveals an astonishing cryptic diversity, which indicates the existence of a species complex with more than 40 cryptic taxa. We therefore suggest a revision of the single species status of E. virens. The phylogeny indicates multiple transitions from diverse sexual ancestor populations to asexuality. Although many transitions appear to be ancient, we argue that this may be an artefact of the existence of unsampled or extinct sexual lineages.

[Screening of modified polyethylene surfaces for tissue engineering of chondrocytes]. / Screening von modifizierten Polyethylenoberflächen für das Tissue-Engineering von Chondrozyten.

BACKGROUND: Reconstructive surgery needs high requirements of substitutes for cartilages. The availability of autologous material is limited. The use of implants can cause inflammatory reactions. Therefore the biocompatibility of porous polyethylene should be improved by masking the synthetic surface with autologous cells. The polyethylene surface was functionalized by collagen, in order to enhance the contact between polyethylene and the surrounding cells. METHODS: The modified surfaces were characterized and tested by an in vitro screening with primary human chondrocytes. RESULTS: The modification of polyethylene surfaces by collagen coating increased the life time of chondrocytes growing at this surface. The effect was independent of the former functionalization. CONCLUSIONS: It is possible to cultivate chondrocytes on polyethylene surfaces. The results have to be proven in a long-term animal experimental study.

No evidence for the 'Meselson effect' in parthenogenetic oribatid mites (Oribatida, Acari).

It has been hypothesized that in ancient apomictic, nonrecombining lineages the two alleles of a single copy gene will become highly divergent as a result of the independent accumulation of mutations (Meselson effect). We used a partial sequence of the elongation factor-1alpha (ef-1alpha) and the heat shock protein 82 (hsp82) genes to test this hypothesis for putative ancient parthenogenetic oribatid mite lineages. In addition, we tested if the hsp82 gene is fully transcribed by sequencing the cDNA and we also tested if there is evidence for recombination and gene conversion in sexual and parthenogenetic oribatid mite species. The average maximum intra-specific divergence in the ef-1alpha was 2.7% in three parthenogenetic species and 8.6% in three sexual species; the average maximum intra-individual genetic divergence was 0.9% in the parthenogenetic and 6.0% in the sexual species. In the hsp82 gene the average maximum intra-individual genetic divergence in the sexual species Steganacarus magnus and in the parthenogenetic species Platynothrus peltifer was 1.1% and 1.2%, respectively. None of the differences were statistically significant. The cDNA data indicated that the hsp82 sequence is transcribed and intron-free. Likelihood permutation tests indicate that ef-1alpha has undergone recombination in all three studied sexual species and gene conversion in two of the sexual species, but neither process has occurred in any of the parthenogenetic species. No evidence for recombination or gene conversion was found for sexual or parthenogenetic oribatid mite species in the hsp 82 gene. There appears to be no Meselson effect in parthenogenetic oribatid mite species. Presumably, their low genetic divergence is due to automixis, other homogenizing mechanisms or strong selection to keep both the ef-1alpha and the hsp82 gene functioning.

Clonal diversity in the ancient asexual ostracod Darwinula stevensoni assessed by RAPD-PCR.

As Darwinulidae (Ostracoda) are considered to be ancient asexuals with a wide geographical and ecological distribution, they are expected to have accumulated mutations during a long timeframe. However, previous studies on genetic variability suggested a low genetic divergence within the darwinulid species Darwinula stevensoni. Here, overall genotopic diversity of D. stevensoni is estimated with the Random Amplified Polymorphic DNA (RAPD) technique. Using six primers revealing 47 consistently scorable polymorphic loci, substantial clonal diversity within this species is detected. Five of the seven surveyed populations are multiclonal. Moreover, the seven populations have a different clonal composition with almost all of the observed clonal genotypes being restricted to single populations, indicating the absence of a single widespread 'clone'. The observed clonal diversity seems to refute the existence of a widespread general purpose genotype for D. stevensoni. However, in light of previously detected uniformity at functional loci, we reconsider the definition of a GPG. We suggest that it need not imply a genome-wide fixed genotype, but rather consists of a set of ecologically relevant genes.

Configuration and racemization determination of cysteine residues in peptides by chiral derivatization and HPLC: application to oxytocin peptides.

An improved RP-HPLC method was developed for the determination of the configuration and stereochemical purity of cysteine residues in peptides. The method consists of oxidation of cysteine and cystine residues to cysteic acid, followed by hydrolysis and pre-column chiral derivatization with Val-Marfey's reagent.

Persistence of asexuality through mixed reproduction in Eucypris virens (Crustacea, Ostracoda).

The ostracod species Eucypris virens exhibits geographical parthenogenesis, with rare sexual populations in southern Europe and widespread asexual populations elsewhere. DNA sequence data from the nuclear ITS1 and mitochondrial COI regions have been used to estimate genetic variabilities and reconstruct phylogenies. The observed divergence was exceptionally high, with intraspecific maxima of 10.3% (ITS1) and 20.9% (COI) among European lineages, levels reported for interspecific comparisons of other taxa. Phylogenetic reconstructions reveal multiple origins of asexual clones from sexual populations. However, we argue that such data can only provide a lower limit on the number of origins of asexual reproduction, and an upper limit on the age of asexual lineages. Congruence between gene trees for different loci can provide support for the inference of long-term apomictic reproduction. Nuclear and mitochondrial data differ in their placement of some asexual clones, possibly indicating that genetic exchange has taken place between sexual and asexual lineages. Such intraspecific hybridization is one route to combine the benefits of both reproductive modes, and it might explain how asexuality managed to persist in E. virens even in long, evolutionary terms.

Regulation of the action of the novel cholecystokinin-releasing peptide diazepam binding inhibitor by inhibitory hormones and taurocholate.

Diazepam binding inhibitor (DBI1-86) has recently been isolated in search for a cholecystokinin (CCK)-releasing peptide in the duodenum that is responsible for the feedback regulation of exocrine pancreatic secretion. Synthetic porcine DBI1-86 stimulates CCK release in vivo and in vitro from isolated intestinal mucosal cells. We postulated that DBI intraduodenally releases CCK in a paracrine fashion and might be the missing link in the feedback regulation of exocrine pancreatic secretion. Somatostatin, peptide YY (PYY) and taurocholate are known to inhibit feedback-stimulated CCK release in the rat. In this study, we investigated the effect of somatostatin, PYY and taurocholate on DBI-stimulated CCK secretion. Dispersed rat intestinal mucosal cells were prepared from the proximal small bowel and continuously perfused. The perfusate was collected and the release of CCK into the medium was measured. DBI1-86 dose-dependently stimulated CCK release, with a maximal effect at 10(-9) M. Somatostatin blocked the DBI-stimulated CCK release. Pretreatment of the cells with pertussis toxin fully reversed the inhibitory effect of somatostatin on DBI-stimulated CCK secretion, suggesting that somatostatin exerts its action by an inhibitory G-protein. In contrast, PYY (10(-6) M) and taurocholate (10(-6) M) did not affect DBI stimulated CCK levels, indicating that they act through different mechanisms to inhibit feedback-stimulated CCK release.

Synthesis of peptide and pseudopeptide amides inhibiting the proliferation of small cell and epithelial types of lung carcinoma cells.

Small cell lung cancer (SCLC) cell lines produce and secrete various peptide hormones, e.g. bombesin (BN)/gastrin releasing peptide (GRP) like peptides that are proposed to function as their autocrine growth factors. To inhibit the proliferative effect of these hormones we have synthesized short chain BN[7-14]-analogues replacing the C-terminal peptide bond by a methylene-amino (-CH2NH-) unit and introducing D-Phe or D-Ser into position 12. As several substance P (SP) analogues were found to inhibit the growth of SCLC cells, some short chain SP-analogues have been synthesized. (Pseudo)octapeptides were synthesized in solution, by fragment condensation using the DCC/HOPfp method. Fragments and SP-analogues were synthesized stepwise using pentafluorophenyl esters. The resistance to hydrolysis of the reduced peptide bond made permitted exact quantification of the Leupsi(CH2NH)Leu pseudopeptide in hydrolysates. The binding ability of both types of peptides to BN-receptors on Swiss 3T3 mouse fibroblast cells and their antiproliferative effect on NCI-H69 human SCLC cell line have been tested and compared with a short chain SP-antagonist pHOPA-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (R) previously described as a potent inhibitor of SCLC proliferation. While BN-analogues showed weak activity in inhibition of proliferation of SCLC cells, SP-analogues 6: D-MePhe-D-Trp-Phe-D-Trp-Leu(psi)(CH2NH)-Leu-NH2 and 7: D-MePhe-DTrp-Phe-D-Trp-Leu-MPA, in spite of greatly diminished affinity towards the BN-receptor, inhibited SCLC proliferation more effectively than R (6: IC50 = 2 microM, 7: IC50 = 5 microM and R: IC50 = 10 microM). Moreover, 6 inhibited the respiratory activity of SK-MES 1 epithelial type of lung carcinoma cells in proliferating but not in the quiescent state, suggesting that the antiproliferative effect of these compounds is not due to simple cytotoxicity. These short chain analogues of SP might be promising candidates as therapeutic agents in the treatment of SCLC.

Diazepam binding inhibitor is a potent cholecystokinin-releasing peptide in the intestine.

Pancreatic proteases in the duodenum inhibit the release of cholecystokinin (CCK) and thus exert feedback control of pancreatic exocrine secretion. Exclusion of proteases from the duodenum either by the diversion of bile-pancreatic juice or by the addition of protease inhibitors stimulates exocrine pancreatic secretion. The mechanism by which pancreatic proteases in the duodenum regulate CCK secretion is unknown. In this study, we isolated a trypsin-sensitive peptide that is secreted intraduodenally, releases CCK, and stimulates pancreatic enzyme secretion in rats. This peptide was found to be identical to the porcine diazepam binding inhibitor by peptide sequencing and mass spectrometry analysis. Intraduodenal infusion of 200 ng of synthetic porcine diazepam binding inhibitor1-86 in rats significantly stimulated pancreatic amylase output. Infusion of the CCK antagonist MK-329 completely blocked the diazepam binding inhibitor-stimulated amylase secretion. Similarly, diazepam binding inhibitor33-52 [corrected] also stimulated CCK release and pancreatic secretion in a dose-dependent manner although it was 100 times less potent than the whole peptide. Using a perfusion system containing isolated mucosal cells from the proximal intestine of rats, porcine diazepam binding inhibitor 10(-12) M) dose dependently stimulated CCK secretion. In separate studies, it was demonstrated that luminal secretion of the diazepam binding inhibitor immunoreactivity (7.5 X 10(11) M) could be detected in rat's intestinal washing following the diversion of bile-pancreatic juice. The secretion of this peptide was inhibited by atropine. In conclusion, we have isolated and characterized a CCK-releasing peptide that has a sequence identical to the porcine diazepam binding inhibitor from pig intestinal mucosa and that stimulates CCK release when administered intraduodenally in rat. This peptide may mediate feedback regulation of pancreatic enzyme secretion.

4-(1-Naphthylamino)-piperidines as inhibitors of lipid peroxidation.

Some analogues of (+/-)-N-methyl-N-¿1-[3-(4-fluorophenoxy)-2-hydroxy-propyl]-piperidin+ ++-4-yl¿ benzothiazol-2-amine (sabeluzole, CAS 104383-17-7), a series of 4-(l-naphthylamino)-piperidines (1a-1h), are potent inhibitors of both NADPH- and Fe(2+)-dependent lipid peroxidation (IC50 < 10 mumol/1). It was demonstrated that the naphthylaminopiperidine moiety is responsible for this effect due to its antioxidant property, verified by cyclic voltammetric studies.

Potentiometric and spectroscopic studies on the copper(II) complexes of peptide hormones containing disulfide bridges.

Copper(II) complexes of oxytocin, 4-Glu-oxytocin, 5-Asp-oxytocin, and GlyGlyGly-Lys8-vasopressin were studied by potentiometric, EPR, and UV-visible spectroscopic methods. The formation of 4N-coordinated complexes was characteristic of all ligands. This type of coordination is especially favored for oxytocin due to the specific conformation of the ring coupled by the disulfide bridge. The coordination of the gamma-carboxylate group of 4-Glu-oxytocin and a disulfide sulfur atom of GlyGlyGly-Lys8-vasopressin was reported to occur in the 2N-complexes over medium pH range.

New short-chain analogs of a substance-P antagonist inhibit proliferation of human small-cell lung-cancer cells in vitro and in vivo.

Human small-cell lung-cancer cells (SCLC) produce and secrete gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin (BN). There is some evidence to suggest that GRP is an autocrine regulator of SCLC cell growth. In the search for potent BN antagonists, several substance-P (SP) analogs were found to inhibit the growth of SCLC cells. We found that a known short-chain SP antagonist, pHOPA-DTrp-Phe-DTrp-Leu-Leu-NH2(NY3238), inhibits the binding of 125I-Tyr4-BN on Swiss 3T3 cell line expressing BN receptors, as well as the proliferation of NCI-H69 SCLC cells. In this study we tested several analogs of NY3238 and we found that NY3521 and NY3460 are more effective in inhibition of proliferation of SCLC cells but less potent in inhibition of binding of 125I-Tyr4-BN on Swiss 3T3 cells than NY3238. Furthermore, we detected specific binding of radiolabelled NY3238 even below 1 nM on NCI-H69 cells that could have been inhibited by SP and NY3460 rather than by BN. In addition to these in vitro studies, NY3460 proved to be effective in inhibiting the growth of NCI-H69 SCLC xenografts in nude mice in vivo. These analogs of NY3238 could be promising therapeutic agents in the treatment of SCLC.

Aspartate-bond isomerization affects the major conformations of synthetic peptides.

The aspartic acid bond changes to an beta-aspartate bond frequently as a side-reaction during peptide synthesis and often as a post-translational modification of proteins. The formation of beta-asparate bonds is reported to play a major role not only in protein metabolism, activation and deactivation, but also in pathological processes such as deposition of the neuritic plaques of Alzheimer's disease. Recently, we reported how conformational changes following the aspartic-acid-bond isomerization may help the selective aggregation and retention of the amyloid beta peptide in affected brains (Fabian et al., 1994). In the current study we used circular dichroism, Fourier-transform infrared spectroscopy, and molecular modeling to characterize the general effect of the beta-aspartate-bond formation on the conformation of five sets of synthetic model peptides. Each of the non-modified, parent peptides has one of the major secondary structures as the dominant spectroscopically determined conformation: a type I beta turn, a type II beta turn, short segments of alpha or 3(10) helices, or extended beta strands. We found that both types of turn structures are stabilized by the aspartic acid-bond isomerization. The isomerization at a terminal position did not affect the helix propensity, but placing it in mid-chain broke both the helix and the beta-pleated sheet with the formation of reverse turns. The alteration of the geometry of the lowest energy reverse turn was also supported by molecular dynamics calculations. The tendency of the aspartic acid-bond isomerization to stabilize turns is very similar to the effect of incorporating sugars into synthetic peptides and suggests a common feature of these post-translational modifications in defining the secondary structure of protein fragments.