Solution structure of the human CD4 (403–419) receptor peptide

The cytoplasmic part of CD4 is known to be essential for the interaction with the human immunodeficiency virus type 1 proteins Vpu and Nef. The 17 amino acid synthetic peptide CD4 (403–419) with the amino acid sequence of the membrane proximal part of the cytoplasmic domain of the human CD4 receptor was structurally investigated by circular dichroism and nuclear magnetic resonance spectroscopy. The average -helical content of the peptide could be estimated to be around 25%. Chemical shift index analysis and the connectivity pattern in nuclear Overhauser enhancement spectra located the -helical part of the peptide from Gln403 to Arg412. It may be speculated that this amphipathic -helix is the contact region with the Vpu and Nef proteins.The authors thank Prof. F.X. Schmid for help with the CD spectra.     

The effects of river-level oscillations on the macroinvertebrate community in a river–floodplain system

Limnology - Extreme climatic events, such as flooding and drought, can abruptly modify the amplitude of the river level of a river, promoting new environmental conditions and impacting aquatic...     

In vivo effects of N/OFQ(1–13)NH2 and its structural analogue [ORN9]N/OFQ(1–13)NH2 on carrageenan-induced inflammation: rat-paw oedema and antioxidant status

The effects of nociceptin(1–13)NH₂ (N/OFQ(1–13)NH₂) and its structural analogue [Orn⁹]N/OFQ(1–13)NH₂ on acute carrageenan (CG)-induced peripheral inflammation and paw antioxidant status were studied. CG was injected intraplantarly in the right hind paw of rats and the volume of the inflamed paw was measured each 30 min for a period of 4h. When administered simultaneously with CG, N/OFQ(1–13)NH₂ decreased the paw volume, whereas if injected 15 min before CG it had no effect. [Orn₉]N/OFQ(1–13)NH₂ produced the opposite effects at the same time-intervals of its administration. We also investigated whether these neuropeptides influence CG-induced changes in cell antioxidant system, especially at the 4th hour of CG administration. CG alone decreased the glutathione level and superoxide dismutase activity, as measured in post-nuclear homogenate of the inflamed paw. However, CG injection increased glutathione peroxidase and glucose-6-phospate dehydrogenase activities, while the activity of glutathione reductase was unchanged. The peptides themselves did not change all measured parameters. Moreover, neither N/OFQ(1–13)NH₂ nor [Orn⁹]N/OFQ(1–13)NH₂ modified CG-induced changes in the antioxidant status, regardless of the time of their injection (simultaneously or 15 min before CG). The present results suggest that N/OFQ(1–13)NH₂ and [Orn⁹]N/OFQ(1–13)NH₂ most likely affect the neuronal inflammation, rather than act as pro- or antioxidants.     

The NOP receptor involvement in both withdrawal- and CCk-8-induced contracture responses of guinea pig isolated ileum after acute activation of κ-opioid receptor

In isolated guinea-pig ileum (GPI), the κ-opioid acute withdrawal response is under the control of several neuronal signaling systems, including the μ-opioid, the A₁-adenosine and the CB₁ receptors, which are involved in the inhibitory control of the κ-withdrawal response. After κ-opioid system stimulation, indirect activation of μ-opioid, A₁-adenosine and CB₁ systems is prevented by the peptide cholecystokinin-8 (CCk-8). In the present study, we have investigated whether the NOP system is also involved in the regulation of the acute κ-withdrawal response. Interestingly, we found that in GPI preparation, the NOP system is not indirectly activated by the κ-opioid receptor stimulation, but instead this system is able by itself to directly regulate the acute κ-withdrawal response. Specifically, our results clearly highlight first the existence of an endogenous tone of the NOP system in GPI, and second that it behaves as a functional anti-opioid system. We also found that, the NOP receptor system is involved in the regulation of the CCk-8-induced contracture intensity, only when in the presence of the κ-opioid receptor stimulation. This effect seems to be regulated by an activation threshold mechanism. In conclusion, the NOP system could act as neuromodulatory system, whose action is strictly related to the modulation of both excitatory and inhibitory neurotransmitters released in GPI enteric nervous system.     

The transformation of the cytoplasmic oestradiol–receptor complex into the nuclear complex in a uterine cell-free system

Experiments performed with a cell-free system in tris-EDTA buffer, pH 7.4, indicate that the high-speed supernatant fraction of the rat uterus contains all the factors necessary to transform the 8S cytoplasmic oestradiol-receptor complex to the nuclear complex. The transformation is temperature-dependent. This nuclear complex was extracted in the form of a 5S particle with 0.4m-KCl from sediments of either uterine or heart nuclei that had been incubated together with the cytoplasmic soluble fraction of the uterus at 2 degrees C for 30min. This complex can also be obtained similarly from the soluble fraction of the uterus, incubated in the absence of nuclei. Previous warming of the soluble fraction to 37 degrees C for 7min was necessary for the successful extraction of the nuclear particle under these conditions of incubation. After an incubation of the transformed complex with the nuclear sediment at 37 degrees C for 7min, the 5S complex was extractable from the uterine nuclear sediment but not from the heart nuclear sediment, which may indicate the tissue specificity of the nuclear acceptor sites for the transformed complex. The extracted uterine nuclear complex sediments in the 5S region, but whether it is the native complex or a subunit or other part of the native complex resulting from the extraction with salt is unknown.     

Common variations in noncoding regions of the human natriuretic peptide receptor A gene have quantitative effects

Genetic susceptibility to common conditions, such as essential hypertension and cardiac hypertrophy, is probably determined by various combinations of small quantitative changes in the expression of many genes. NPR1, coding for natriuretic peptide receptor A (NPRA), is a potential candidate, because NPRA mediates natriuretic, diuretic, and vasorelaxing actions of the nariuretic peptides, and because genetically determined quantitative changes in the expression of this gene affect blood pressure and heart weight in a dose-dependent manner in mice. To determine whether there are common quantitative variants in human NPR1, we have sequenced the entire human NPR1 gene and identified 10 polymorphic sites in its non-coding sequence by using DNA from 34 unrelated human individuals. Five of the sites are single nucleotide polymorphisms; the remaining five are length polymorphisms, including a highly variable complex dinucleotide repeat in intron 19. There are three common haplotypes 5' to this dinucleotide repeat and three 3' to it, but the 5' haplotypes and 3' haplotypes appear to be randomly associated. Transient expression analysis in cultured cells of reporter plasmids with the proximal promoter sequences of NPR1 and its 3' untranslated regions showed that these polymorphisms have functional effects. We conclude that common NPR1 alleles can alter expression of the gene as much as two-fold and could therefore significantly affect genetic risks for essential hypertension and cardiac hypertrophy in humans.     

Synthesis and biological activity of new series of N-modified analogues of the N/OFQ(1–13)NH2 with aminophosphonate moiety

New series of N-modified analogues of the N/OFQ(1-13)NH(2) with aminophosphonate moiety have been synthesized and investigated for biological activity. These peptides were prepared by solid-phase peptide synthesis-Fmoc-strategy. The N/OFQ(1-13)NH(2) analogues were tested for agonistic activity in vitro on electrically stimulated rat vas deferens smooth-muscle preparations isolated from Wistar albino rats. Our study has shown that the selectivity of the peptides containing 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids to the N-side of Phe is not changed-they remain selective agonists of NOP receptors. The derivative with the largest ring (NOC-6) demonstrated efficacy similar to that of N/OFQ(1-13)NH(2), but in a 10-fold higher concentration. The agonistic activity of newly synthesized N-modified analogues of N/OFQ(1-13)NH(2) with aminophosphonate moiety was investigated for the first time.     

Structure–function analysis of VEGF receptor activation and the role of coreceptors in angiogenic signaling

Vascular endothelial growth factors (VEGFs) constitute a family of six polypeptides, VEGF-A, -B, -C, -D, -E and PlGF, that regulate blood and lymphatic vessel development. VEGFs specifically bind to three type V receptor tyrosine kinases (RTKs), VEGFR-1, -2 and -3, and to coreceptors such as neuropilins and heparan sulfate proteoglycans (HSPG). VEGFRs are activated upon ligand-induced dimerization mediated by the extracellular domain (ECD). A study using receptor constructs carrying artificial dimerization-promoting transmembrane domains (TMDs) showed that receptor dimerization is necessary, but not sufficient, for receptor activation and demonstrates that distinct orientation of receptor monomers is required to instigate transmembrane signaling. Angiogenic signaling by VEGF receptors also depends on cooperation with specific coreceptors such as neuropilins and HSPG. A number of VEGF isoforms differ in binding to coreceptors, and ligand-specific signal output is apparently the result of the specific coreceptor complex assembled by a particular VEGF isoform. Here we discuss the structural features of VEGF family ligands and their receptors in relation to their distinct signal output and angiogenic potential.     

The ontogeny of components of the renin–angiotensin system in the porcine fetal ovary

There is an autonomous renin–angiotensin system (RAS) in the adult ovary. Renin is present in the primitive kidney, and the fetal ovary develops from the nephrogenic ridge. We hypothesised that components of the ovarian RAS would be present from early gestation, with potential roles in ovarian development. We studied fetal pig ovaries from approximately day 45 (～0.39 gestation) to term and measured mRNA (RT-PCR) for prorenin, angiotensinogen and the angiotensin II (AngII) Type 1 and 2 receptors (AT₁ and AT₂), and protein expression (Western blot) and localization (immunohistochemistry) of the AT₁ and AT₂ receptors. mRNA for prorenin was present in relatively low abundance from at least day 45 and rose to ～day 75 of gestation, whilst mRNA for angiotensinogen rose steadily. mRNA for the AT₁ receptor was present from approximately day 45 and did not alter significantly with increasing gestation but AT₂ receptor mRNA was initially high, falling sharply through pregnancy. The AT₁ receptor protein abundance fell steadily to term, whereas the AT₂ receptor protein did not change during gestation. Both receptors were localised in the surface epithelium and egg nests, the granulosa cells of primordial, primary and secondary follicles, and the oocytes of all except the secondary follicles. Collectively, our results support the hypothesis that there is a functional RAS in the fetal ovary from at least approximately day 45 of gestation until term and that it may have a paracrine role in ovarian growth and development.     

Immunohistochemical localization of the angiotensin-(1–7) receptor Mas in the murine forebrain

Apart from the well-known biologically active angiotensin II, other biologically active angiotensins have been discovered, including angiotensin IV and angiotensin-(1–7). Some years ago, we and others discovered that the Mas proto-oncogene encodes a receptor that is essential for angiotensin-(1–7) signaling. Angiotensin-(1–7) is not only expressed in the periphery but also within the brain. Based on that, we examined the distribution of Mas within the murine brain, using an antibody directed against the 3^rd cytoplasmic loop of the receptor protein. Strongest Mas protein expression was detected in the dentate gyrus of the hippocampus and within the piriform cortex. However, Mas protein expression is not restricted to these areas, since Mas immunopositive neurons were also seen in different parts of the cortex, hippocampus, amygdala, basal ganglia, thalamus and hypothalamus. Based on the expression of Mas protein in the cortex and the limbic system, angiotensin-(1–7) signaling may play a role in synaptic plasticity, learning, memory and emotion, as has been described for angiotensin II and IV.     

The presence of N(ε)-(Carboxymethyl) lysine in the human epidermis

It is well known that advanced glycation end products (AGEs) are formed in long-lived dermal proteins such as collagen, and that their formation is related to skin aging. To examine the distribution of AGEs in skin tissue, we performed immunofluorescence studies on the human skin using an anti-AGEs antibody. Interestingly, AGEs signals were observed not only in the dermis but also in the epidermis. The objectives of this study were to confirm the presence of N(ε)-(Carboxymethyl) lysine (CML), an AGE structure, in the epidermis and to characterize the CML-modified proteins. The presence of CML in the stratum corneum (SC) was examined using liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Concordance between the retention times of a compound in the SC hydrolysate and authentic CML, as well as with the specific mass transition of CML, was detected. This result showed that CML is present in the epidermis. In order to characterize the CML-modified proteins in the epidermis, protein samples extracted from the SC were analyzed using two-dimensional electrophoresis followed by an amino acid sequence analysis. The clarified peptide sequences covered approximately 27% of the amino acid sequences of cytokeratin 10 (K10). In the immunoblotting experiment following the two-dimensional electrophoresis, where protein samples extracted from whole epidermis were used, the position of the major CML-positive spots corresponded to those of K10. Taken together these results showed that CML is present in the human epidermis, and suggest that K10 is one of the target molecules for CML modification in the epidermis.     

Evaluation of hydrogen bonds of ecdysteroids in the ligand–receptor interactions using a protein modeling system

The insect molting hormone, 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) specifically bind to the ecdysone receptor. Previously, we synthesized various ecdysteroids containing the side chain moiety of ponasterone A (PonA), and measured the binding activity against Drosophila Kc cells to study the structure–activity relationship. Here we quantitatively analyzed the structure–activity relationship for the ligand binding of ecdysteroids including 20E and PonA. Since the hydrogen bonding (HB) is one of the important physicochemical properties for ligand binding to the ecdysteroid receptor, the number of possible HBs between the ligand molecule and the receptor was manually counted in the modeled ligand–receptor complex for all compounds. The construction of the ligand–receptor model was executed by the full-automatic modeling system (FAMS) in which calculation was done by simulated annealing. The binding potency of 15 ecdysteroids to Kc-cells were linearly correlated (r² = 0.63) with the number of HBs which are observed between ligand and receptor molecule. Contribution of steric and electrostatic effects on the ligand–receptor binding was also examined using a three-dimensional quantitative structure–activity relationship (3-D QSAR), comparative molecular field analysis (CoMFA).     

The long-term (1979–2005) effects of the North Atlantic Oscillation on wind-induced wave mixing in Loch Leven (Scotland)

We report on long-term covariation (1979–2005) between indices of the North Atlantic Oscillation (NAO) and wind speed and direction in Loch Leven. The effects of the observed variations in wind speed and direction were combined to produce modelled wave-mixed depths (Zc). Positive correlations were observed between seasonal and annual wind speeds and westerly frequency and indices of the NAO that are in line with general perception: positive NAO was correlated with stronger, more westerly winds and these correlations were strongest in winter and spring. Correlations between NAO and estimates of Zc were strongest in the most westerly exposed site in spring (r ² = 0.701; Zc_spring versus spring NAO index). On average, over a 25-year period Zc was deeper in spring and shallower in summer. Major anomalies from the 25-year seasonal means were observed in 1982, 1979, and 1991. Annual average Zc was low in the late 1970s and early 1980s (shallowest average annual Zc of 1.0 m (1984)), high in the late 1980s and early 1990s (deepest average annual Zc of 1.9 m (1990)) and moderate in recent years (up to 2005). This study has major implications for our understanding of potential climate change drivers and the related responses of shallow lake ecosystems, including alterations to littoral habitat quality and benthic–pelagic coupling.