Tetrahydrobiopterin restores endothelial dysfunction induced by an oral glucose challenge in healthy subjects

An oral glucose challenge causes transient impairment of endothelial function, probably because of increased oxidative stress. During oxidative stress, endothelial nitric oxide (NO) synthase (eNOS) becomes uncoupled because of decreased bioavailability of tetrahydrobiopterin (BH4), an essential cofactor of eNOS. Therefore, we examined whether an acute supplement of BH4 could restore endothelial dysfunction induced by an oral glucose challenge. Healthy subjects were examined in 53 experiments. Forearm blood flow was measured by venous occlusion plethysmography. Dose-response studies were obtained during intra-arterial infusion of serotonin to elicit endothelium-dependent, NO-specific vasodilation and during sodium nitroprusside (SNP) infusion to elicit endothelium-independent vasodilation. Subjects were examined before (fasting) and 1 and 2 h after an oral glucose challenge (75 g) with serotonin (n = 10) and SNP (n = 8). On different days (6R)-5,6,7,8-tetrahydro-l-biopterin dihydrochloride (6R-BH4; n = 10), the active cofactor of eNOS or its stereoisomer (6S)-5,6,7,8-tetrahydro-l-biopterin sulfate (6S-BH4; n = 10), which is inactive as a cofactor, was added 10 min (500 microg/min) before and during the 1-h postchallenge serotonin dose-response study. In vitro studies showed that 6R-BH4 and 6S-BH4 were equipotent antioxidants. Serotonin response was reduced by 24 +/- 7% (at the highest dose) at 1 h postchallenge compared with fasting (P = 0.001) and was restored 2 h postchallenge. The reduction was reversed by the administration of 6R-BH4 but not by 6S-BH4. SNP responses were slightly increased 1 and 2 h postchallenge (increased by 15 +/- 13% at third dose 2 h postchallenge, P = 0.0001). An oral glucose challenge causes transient, NO-specific, endothelial dysfunction, which may be reversed by BH4. Transient postprandial endothelial dysfunction may be partly explained by reduced bioavailability of BH4 and NO.     

Further structures in the jaw apparatus of Limnognathia maerski (Micrognathozoa), with notes on the phylogeny of the Gnathifera

The jaws of Limnognathia maerski, Micrognathozoa, were investigated with light- and scanning electron microscopy. The study yielded several new structures and sclerites, including the ventral part of main jaw, the pharyngeal lamellae, the manus, the dorsal and ventral fibularium teeth, and a reinterpretation of the fibularium compartmentalization. Furthermore, it was shown that several jaw elements are composed of densely packed rods. Comparison with Rotifera and Gnathostomulida suggested that the micrognathozoan main jaw is homologous with the rotifer incus and the gnathostomulid articularium and that the pseudophalangids (the ventral jaws) and their associated sclerites correspond to the rotifer mallei. These results imply that Micrognathozoa is more closely related to Rotifera than to Gnathostomulida.     

Cyclooxygenase-2 contributes to elevated renin in the early postnatal period in rats

We asked whether cyclooxygenase (COX) activity controls the renin-angiotensin system in the postnatal period. During kidney development, renin peaked at postnatal days 0-1 at the mRNA, tissue protein [renal renin concentration (RRC)], and plasma renin concentration (PRC) levels and was widely expressed along preglomerular vessels. PRC and renin mRNA expression was elevated until weaning in the 4th postnatal week compared with adult rats. Renocortical COX-2 was restricted to Tamm-Horsfall protein-positive cells in the thick ascending limb of Henle's loop, and cortical COX-2 mRNA and protein expression were elevated along with PRC in the 2nd and 3rd postnatal weeks. In contrast, cortical COX-1 expression was constant, but medullary COX-1 expression increased eightfold from the 1st to 4th postnatal week. A COX-2-selective blocker, parecoxib, and a nonselective blocker, indomethacin, given in a period with COX-2 induction from postnatal day 6 to day 12, markedly decreased PRC, but not renin mRNA or RRC. Inhibition of angiotensin AT(1) receptors by candesartan from postnatal day 1 to day 5 increased COX-2 mRNA (2.5-fold), protein, and distribution, renin mRNA (7-fold) and PRC (20- to 70-fold), but had no influence on COX-1 mRNA. Thus, due to very low levels of expression, COX-2 is unlikely to be responsible for the birth peak of renin, but COX-2 activity supports renin secretion later in the suckling period. ANG II negatively feeds back on renocortical COX-2 expression in the 1st postnatal days with high activity of the renin system. We suggest that suckling in the rat is correlated to an enhanced, COX-2-mediated, secretory activity of renin-producing juxtaglomerular cells.     

Fluid pressure in human dermal fibroblast aggregates measured with micropipettes

Previous studies indicated that connective tissue cells in dermis are involved in control of interstitial fluid pressure (P_if). We wanted to develop and characterize an in vitro model representative of loose connective tissue to study dynamic changes in fluid pressure (P_f) over a time course of a few minutes. P_f was measured with micropipettes in human dermal fibroblast cell aggregates of varying size (<100- and >100-µm diameter) and age (days 1-4) kept at different temperatures (15, 25, and 35°C). Pressures were measured at different depths of micropipette penetration and after treatment with prostaglandin E₁ isopropyl ester (PGE₁), latanoprost (PGF₂), and ouabain. P_f was positive (more than +2 mmHg) during control conditions and increased with increasing aggregate size (day 2), age (day 4 vs. day 1), temperature, and depth of micropipette penetration. P_f decreased from 2.9 to 2.0 mmHg during the first 10 min after application of 10 µl of 1 mM PGE₁ (P < 0.001). P_f increased from 3.0 to 4.8 mmHg (P < 0.01) after administration of 10 µl of 1.4 µM ouabain and from 3.1 to 4.4 mmHg after addition of 5 µl of 1.42 mM PGF₂ (P > 0.05). In conclusion, we have developed and validated a new in vitro method for studying fluid pressure in loose connective tissue elements with the advantage of allowing reliable and rapid screening of substances that have a potential to modify P_f and studying in more detail specific cell types involved in control of P_f. This study also provides evidence that fibroblasts in the connective tissue can actively modulate P_f. micropuncture; prostaglandin E₁; prostaglandin F₂; ouabain; integrins     

Evidence of a hybrid-zone in Atlantic cod (Gadus morhua) in the Baltic and the Danish Belt Sea revealed by individual admixture analysis

The study of hybrid zones is central to our understanding of the genetic basis of reproductive isolation and speciation, yet very little is known about the extent and significance of hybrid zones in marine fishes. We examined the population structure of cod in the transition area between the North Sea and the Baltic Sea employing nine microsatellite loci. Genetic differentiation between the North Sea sample and the rest increased along a transect to the Baltic proper, with a large increase in level of differentiation occurring in the Western Baltic area. Our objective was to determine whether this pattern was caused purely by varying degrees of mechanical mixing of North Sea and Baltic Sea cod or by interbreeding and formation of a hybrid swarm. Simulation studies revealed that traditional Hardy-Weinberg analysis did not have sufficient power for detection of a Wahlund effect. However, using a model-based clustering method for individual admixture analysis, we were able to demonstrate the existence of intermediate genotypes in all samples from the transition area. Accordingly, our data were explained best by a model of a hybrid swarm flanked by pure nonadmixed populations in the North Sea and the Baltic Sea proper. Significant correlation of gene identities across loci (gametic phase disequilibrium) was found only in a sample from the Western Baltic, suggesting this area as the centre of the apparent hybrid zone. A hybrid zone for cod in the ecotone between the high-saline North Sea and the low-saline Baltic Sea is discussed in relation to its possible origin and maintenance, and in relation to a classical study of haemoglobin variation in cod from the Baltic Sea/Danish Belt Sea, suggesting mixing of two divergent populations without interbreeding.     

Phylogeography of Cavernularia hultenii: evidence of slow genetic drift in a widely disjunct lichen

Population structure and history is poorly known in most lichenized ascomycetes. Many species display large-scale infraspecific disjunctions, which have been explained alternately by range fragmentation in species of high age and widespread long-distance dispersal. Using the lichen Cavernularia hultenii, which is widely disjunct across North America and Europe, Pleistocene and Holocene population history was inferred. The internal transcribed spacer (ITS) and part of the the intergenic spacer (IGS) region of the nuclear ribosomal DNA were sequenced in 300 individuals representing 62 populations across the range of the species. While four ancestral haplotypes are found in all areas, none of the observed tip haplotypes is present in more than one of the three part ranges. Although this is evidence for a past fragmentation event, nested clade analysis (NCA) remains equivocal in the choice between allopatric fragmentation and long-distance dispersal. Mismatch distributions indicate exponential population growth, probably during postglacial invasion of C. hultenii into formerly glaciated areas of western North America. The presence of one southern and at least one northern glacial refugium in South Central Alaska is inferred. Evidence for another refugium in the Queen Charlotte Islands or Alexander Archipelago is inconclusive because of sparse sampling. However, a range expansion was not confirmed unambiguously by NCA. The limited power of NCA to infer past range fragmentations and expansions is due apparently to the shallow haplotype network and widespread ancestral haplotypes. This can be explained by slow genetic drift causing incomplete removal of ancestral haplotypes from the postfragmentation and postexpansion areas.     

How big is the iceberg of which organellar genes in nuclear genomes are but the tip?

As more and more complete bacterial and archaeal genome sequences become available, the role of lateral gene transfer (LGT) in shaping them becomes more and more clear. Over the long term, it may be the dominant force, affecting most genes in most prokaryotes. We review the history of LGT, suggesting reasons why its prevalence and impact were so long dismissed. We discuss various methods purporting to measure the extent of LGT, and evidence for and against the notion that there is a core of never-exchanged genes shared by all genomes, from which we can deduce the "true" organismal tree. We also consider evidence for, and implications of, LGT between prokaryotes and phagocytic eukaryotes.     

A kinetic study on iron stimulation of the xanthine oxidase dependent oxidation of ascorbate

Xanthine oxidase reduces molecular oxygen to H₂O₂ and superoxide radicals during its catalytic action on xanthine, hypoxanthine or acetaldehyde. Ascorbate is catalytically oxidized by the superoxide radicals generated, when present in the reaction solution (Nishikimi 1975). The present study shows that iron ions markedly stimulate the enzyme dependent ascorbate oxidation, by acting as a red/ox-cycling intermediate between the oxidase and ascorbate. An apparent K_m-value of 10.8 M characterized the iron stimulatory effect on the reaction at pH 6.0. Reduced transition-state metals can be oxidized by H₂O₂ through a Fenton-type reaction. Catalase was found to reduce the effect of iron on the enzyme dependent ascorbate oxidation, strongly suggesting that H₂O₂, produced during catalysis, is involved in the oxidation of ferrous ions.     

Effect of 3-5 monocyclizations of angiotensin II and 4-aminoPhe6-Ang II on AT2 receptor affinity

The endogenous angiotensin II (Ang II) and the synthetic AT(2) selective agonist 4-aminoPhe(6)-Ang II respond very differently to identical cyclizations. Cyclizations of Ang II by thioacetalization, involving the 3 and 5 amino acid residue side chains, provided ligands with almost equipotent binding affinities to Ang II at the AT(2) receptor. In contrast, the same cyclization procedures applied on the AT(2) selective 4-aminoPhe(6)-Ang II delivered significantly less potent AT(2) receptor ligands, although the AT(2)/AT(1) selectivity was still very high. The fact that different structure-activity relationships are observed after imposing conformational restrictions on Ang II and 4-aminoPhe(6)-Ang II, respectively, suggests that the peptides, despite large similarities might adopt quite different backbone conformations when binding to the AT(2) receptor.     

HER-2 DNA and protein vaccines containing potent Th cell epitopes induce distinct protective and therapeutic antitumor responses in HER-2 transgenic mice

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in approximately 20-30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule. Vaccination with HER-2 DNA protected nontransgenic mice from tumor challenge, but induced only moderate protection in one of the tumor models. However, vaccination with the modified HER-2 protein resulted in almost complete protection from tumor challenge in both tumor models. This protection could be mediated by Abs alone. In addition, protein vaccination efficiently eliminated pre-established tumors in both models, even when vaccination occurred 9 days after tumor implantation. These data demonstrate the potential of HER-2-based vaccines as therapeutic agents for the treatment of cancers overexpressing HER-2.