Internalization of [3H]substance P analogues in NK-1 receptor transfected CHO cells

The internalization of [3H]propionyl[Met(O2)11]SP(7-11) which binds one binding site and of [3H][Pro9]SP which binds the two binding sites associated with the NK-1 receptor has been examined in CHO cells. The quantity of [3H][Pro9]SP measured inside the cytoplasm in kinetic experiments is fully temperature-dependent. In contrast, [3H]propionyl[Met(O2)11]SP(7-11) internalization reaches the same extent whatever the temperature, although the rate slowed down with lower temperature. The extent of internalization of [3H][Pro(9)]SP relative to the total specific bound is biphasic, when the extent of internalization of [3H]propionyl[Met(O2)11]SP(7-11) remains constant. For [3H][Pro9]SP, a high-affinity high-yield component inhibited in the presence of propionyl[Met(O2)11]SP(7-11) and a low-affinity low-yield component in the internalization process could be determined. Saturation studies show that [3H][Pro9]SP-binding parameters are insensitive to both phenylarsine oxide and monensin treatment, whereas [3H]propionyl[Met(O2)11]SP(7-11) maximal binding is decreased in both cases. Altogether, these data suggest that the two radiolabeled peptides should not follow the same internalization pathway.     

Involvement of the second extracellular loop (E2) of the neurokinin-1 receptor in the binding of substance P. Photoaffinity labeling and modeling studies

Substance P (SP) interacts with the neurokinin-1 (NK-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine ((p-Bz)Phe) in position 8 of their sequence. This study was carried out with [BAPA-Lys(6),(p-Bz)Phe(8),Pro(9),Met(O(2))(11)]SP-(7-11) and [BAPA(0),(p-Bz)Phe(8)]SP on both rat and human NK-1 receptors expressed in CHO cells. Combined trypsin and endo-GluC enzymatic complete digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis led to the identification of the same domain of covalent interaction, (173)TMPSR(177), for the two photoactivatable peptides. Further digestion of this fragment with carboxypeptidase Y led to the identification of (173)TMP(175) in the second extracellular loop (E2) of the NK-1 receptor as the site of covalent attachment. Models of the conformation of this E2 loop in the human NK-1 receptor were generated using two different strategies, one based on homology with bovine rhodopsin and the other based on the solution conformation preferences of a synthetic peptide corresponding to the E2 loop.     

Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1

Despite the importance of the stress-activated protein kinase pathways in cell death and survival, it is unclear how stressful stimuli lead to their activation. In the case of heat shock, the existence of a specific mechanism of activation has been evidenced, but the molecular nature of this pathway is undefined. Here, we found that Ask1 (apoptosis signal-regulating kinase 1), an upstream activator of the stress-activated protein kinase p38 during exposure to oxidative stress and other stressful stimuli, was also activated by heat shock. Ask1 activity was required for p38 activation since overexpression of a kinase dead mutant of Ask1, Ask1(K709M), inhibited heat shock-induced p38 activation. The activation of Ask1 by oxidative stress involves the oxidation of thioredoxin, an endogenous inhibitor of Ask1. A different activation mechanism takes place during heat shock. In contrast to p38 induction by H(2)O(2), induction by heat shock was not antagonized by pretreatment with the antioxidant N-acetyl-l-cysteine or by overexpressing thioredoxin and was not accompanied by the dissociation of thioredoxin from Ask1. Instead, heat shock caused the dissociation of glutathione S-transferase Mu1-1 (GSTM1-1) from Ask1 and overexpression of GSTM1-1-inhibited induction of p38 by heat shock. We concluded that because of an alternative regulation by the two distinct repressors thioredoxin and GSTM1-1, Ask1 constitutes the converging point of the heat shock and oxidative stress-sensing pathways that lead to p38 activation.     

Pharmacological characterization of tachykinin septide-sensitive binding sites in the rat submaxillary gland

Binding studies have shown that [125I]NKA is a selective ligand of tachykinin septide-sensitive binding sites from membranes of the rat submaxillary gland. Indeed, this ligand bound with high affinity to a single population of sites. In addition, competition studies indicated that natural tachykinins and tachykinin-related compounds had a similar affinity for these sites than for those labeled with [3H]ALIE-124, a selective ligand of septide-sensitive binding sites. Moreover, selective tachykinin NK2, or NK3 agonists or antagonists exhibited weak or no affinity for [125I]NKA binding sites. As indicated by Ki values of several compounds, the pharmacological characteristics of the septide-sensitive binding sites (labeled with [125I]NKA) largely differ from those of classic NK1 binding sites, as determined on crude synaptosomes from the rat brain using [125I]Bolton-Hunter substance P (SP) as ligand. Indeed, several tachykinins including neurokinin A (NKA), neuropeptide K (NPK), neuropeptide gamma (NKgamma), and neurokinin B, as well as some SP and NKA analogues or C-terminal fragments such as septide, ALIE-124, SP(6-11), NKA(4-10), which have a weak affinity for classic tachykinin NK1 binding sites exhibited a high affinity for the septide-sensitive binding sites. In contrast, SP, classic selective NK1 agonists, and antagonists had a high affinity for both types of binding sites. The presence of a large population of tachykinin septide-sensitive binding sites in the rat submaxillary gland may thus explain why NPK and NPgamma induce salivary secretion and may potentiate the SP-evoked response in spite of the absence of tachykinin NK2 receptors in this tissue.     

Adventitious shoot production from strips of stem in the Dutch elm hybrid ‘Commelin’: plantlet regeneration and neomycin sensitivity

The regenerative ability of small strips of stem of the Dutch elm hybrid ‘Commelin’ was tested as well as its sensitivity to neomycins. Cambium explants (1 mm thick), were excised from woody stems collected in the field. Up to 20 buds/explant were induced within 2–3 weeks giving 2–5 rootable shoots/explant after 5–6 weeks. Shoot excision every week from week three improved the yield up to 7 shoots per explant. Fourteen and 2.9 μM GA3 promoted shoot growth. Cytokinins (1 μM zeatin or 5 μM BA or 0.05 μM TDZ) completely inhibited shoot production and promoted callus formation. Kanamycin and paromomycin at between 240 and 360 μM inhibited shoot formation as did geneticin at 80 μM. The shoot-forming ability of the explants was high from leaf fall in the autumn to the spring flush, but could be maintained up to September by using cold storage (5–7 °C). Ninety-six percent of the shoots rooted with 0.5 μM IBA and were successfully acclimatized despite having a large basal callus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Augmentation of transmidline skin perfusion and viability in transverse rectus abdominis myocutaneous (TRAM) flaps in the pig.

The aim of this experiment was to design a clinically relevant TRAM flap in the pig and to use this flap model to study the effectiveness of preoperative ligation of the dominant vascular pedicle in augmentation of muscle and skin capillary blood flow and skin viability in the TRAM flap. This TRAM flap model was based on the deep inferior epigastric vascular pedicle, with the center of the transverse skin paddle attached to the underlying rectus abdominis muscle at the superior end of the muscle and extending bilaterally from its attached muscle. The transverse skin paddle (8 x 30 cm) included a contralateral and ipsilateral random portion of skin. This flap model was based on the deep inferior epigastric rather than the superior epigastric vascular pedicle because the deep inferior epigastric vascular pedicle is the smaller of the two in the pig and augmentation of its blood supply by ligation of the dominant superior epigastric vascular pedicle resembles more closely the clinical situation. It was observed that ligation of the dominant superior epigastric vascular pedicle 14 days prior to raising the TRAM flap significantly (p less than 0.05; n = 5) increased the total muscle and skin capillary blood flow and skin viability in the transverse skin paddle compared with the sham-operated control (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

The aspartic acid in deltorphin I and dermenkephalin promotes targeting to delta-opioid receptor independently of receptor binding.

Recent studies on the highly potent and selective delta-opioid agonists demenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) suggested that key structural features necessary for specific targetting to the delta-opioid receptor are located within the C-terminal halves of these naturally occurring heptapeptides. To investigate the contribution of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin to the delta-addressing ability of the C-terminal ends, fourteen analogs were synthesized and assessed for their ability to bind to mu and delta-opioid receptors in rat brain membrane homogenates. Results showed that i/ although the tetrapeptide C-terminus of dermenkephalin and deltorphin I differ in amino acid composition, they play a similar role in specifying correct addressing of these peptides to the delta-receptor, ii/ the negatively charged side chain of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin is not involved in binding contact at the delta-receptor site, nor in maintaining a delta-bioactive folding of the peptides, iii/ these side chains are, in contrast, functionally or structurally required to confer high delta-selectivity by preventing mu-site recognition and/or binding.     

Stabilized recombinant suppressors of RNA silencing: functional effects of linking monomers of Carnation Italian Ringspot virus p19

Eukaryotes have evolved complex cellular responses to double-stranded RNA including the RNA silencing pathway. Tombusviruses have adapted a mechanism to evade RNA silencing that involves a 19 kDa dimeric protein (p19) that is a suppressor of RNA silencing. In order to develop stabilized p19 proteins, linked versions of p19 from the Carnation Italian Ringspot virus (CIRV) were constructed that joined the C-terminus of one subunit to the N-terminus of the second subunit. Like the native CIRV p19, these linked p19 proteins were able to bind to double-stranded siRNAs with nanomolar affinity and discriminate siRNA according to length. In addition, the interdomain linker improved both the stability and binding properties of the p19 dimer. The observed binding properties support the idea that the semi-rigid cross-link favors the folded, binding-competent state of p19. The cross-linked recombinant CIRV-p19s represent novel stabilized suppressors of RNA silencing and may be useful in future biophysical, immunological and cell biology studies.     

A female's past experience with predators affects male courtship and the care her offspring will receive from their father

Differential allocation occurs when individuals adjust their reproductive investment based on their partner''s traits. However, it remains unknown whether animals differentially allocate based on their partner''s past experiences with predation risk. If animals can detect a potential mate''s experience with predators, this might inform them about the stress level of their potential mate, the likelihood of parental effects in offspring and/or the dangers present in the environment. Using threespined stickleback (Gasterosteus aculeatus), we examined whether a female''s previous experience with being chased by a model predator while yolking eggs affects male mating effort and offspring care. Males displayed fewer conspicuous courtship behaviours towards females that had experienced predation risk in the past compared with unexposed females. This differential allocation extended to how males cared for the resulting offspring of these matings: fathers provided less parental care to offspring of females that had experienced predation risk in the past. Our results show for the first time, to our knowledge, that variation among females in their predator encounters can contribute to behavioural variation among males in courtship and parental care, even when males themselves do not encounter a predator. These results, together with previous findings, suggest that maternal predator exposure can influence offspring development both directly and indirectly, through how it affects father care.