An NMR Study of the Conformations of N-terminal Substance P Fragments and Antagonists

Abstract

Three N-terminal fragments of the neurotransmitter Substance P as well as two antagonist heptapeptides containing D-amino-acid residues were studied using different ID and 2D NMR techniques. Total nonexchangeable ¹H-NMR assignments were carried out in D₂O and the NH protons were assigned in H₂O by means of COSY experiments. The spectral data indicates that there are no preferred conformations for the backbone. The N-terminal tetrapeptide SP_1–4-OH exists as a mixture of cis/trans isomers and this effect was studied as a function of pH.     

Bifidobacteria-mediated immune system imprinting early in life

1. Download : Download high-res image (173KB)
2. Download : Download full-size image

     

Technical Factors Affecting an Immunoelectrophoretic Reference System for Analysis of Mycobacterial Antigens

The length of immunoelectrophoretic separation patterns obtained with a reference mycobacterial antigen was reduced significantly when a continuous buffer system was used in place of the recommended discontinuous system. Varying the supporting medium by using 1% concentrations of different agar preparations in the discontinuous system also affected the separation length and resolution of the reference pattern. Measurement of the electrical resistivity of the agar medium provided an explanation of these differences on the basis of current density or field intensity applied per slide. Variation in the reference pattern obtained in differently designed electrophoresis chambers also was attributed to differences in field intensity. To avoid these obvious alterations in the separation pattern of the reference antigen, it was suggested that separation procedures described originally for the reference system be used without modification.     

Modifications des cellules gonadotropes de la préhypophyse chez la ratte injectée de testostérone au cours du cycle oestral

Summary 4-day cyclic adult female Wistar rats were injected subcutaneously with testosterone propionate on diestrus 1 at 16:00 and on diestrus 2 at 10:00 respectively. Non-injected females served as controls. Autopsy was performed on diestrus 2 at 23:00, and on proestrus at 14:00 and 17:00 respectively. The blue Alcian-PAS staining was used to evidence FSH () and LH () pituitary cells.In control animals and in diestrus 2 injected females only a small number of FSH cells could be detected on diestrus 2 at 23:00. This number increased markedly on proestrus at 14:00 and decreased on proestrus at 17:00. A similar evolution was observed in diestrus 1 testosterone injected females, but the number of FSH cells appeared higher at any stage of autopsy in these females than in diestrus 2 injected females and in control rats.In control females, numerous LH cells were observed on diestrus 2 at 23:00. The number of these cells was diminished on proestrus at 14:00 and still more at 17:00. On the contrary few LH cells were detected in testosterone injected females on the evening of diestrus 2. An increase of these cells occurred on proestrus at 14:00, followed at 17:00 by only a weak diminution as established by comparison with control animals.An inhibition of FSH release and a suppression of the proestrus surge of LH were therefore supposed to cause, on one hand, the slowing up of follicular growth observed in diestrus 1 injected females and, on the other hand, the blockage of ovulation noted in both diestrus 1 and diestrus 2 treated animals.

Travail effectué avec l'aide de la D.G.R.S.T. Contrat n^o 72.7.0030.     

Role played by the ovary and adrenals in early receptivity during 4-day cycle in the female rat

The mechanisms involved in the control of precocious sexual receptivity were studied in 4-day cyclic female Wistar rats injected with 10 μg estradiol benzoate (EB) and caged with a male during the night from diestrus II to proestrus. Early mating frequencies were compared in intact females, in animals ovariectomized on the morning of diestrus I, in adrenalectomized and in adrenalectomized-ovariectomized females. No change in early sexual receptivity occurred either in ovariectomized, or in adrenalectomized animals. On the contrary, a significant decrease of precocious mating frequencies was noted in adrenalectomized-ovariectomized females. The role played by the ovary in the control of precocious receptivity was supposed to be due to the secretion of progesterone which has been evidenced on the late afternoon of diestrus II in estrogen treated females.Concerning the mechanisms by which the adrenals may compensate for the ovaries in the control of early sexual receptivity in estrogen-primed females it was observed that notwithstanding an inhibitory action exerted by EB on the adrenal progesterone secretion, a low rate of progesterone was maintained in the peripheral plasma which was compatible with early mating in ovariectomized animals.     

Lung Ischemia: A Model for Endothelial Mechanotransduction

Endothelial cells in vivo are constantly exposed to shear associated with blood flow and altered shear stress elicits cellular responses (mechanotransduction). This review describes the role of shear sensors and signal transducers in these events. The major focus is the response to removal of shear as occurs when blood flow is compromised (i.e., ischemia). Pulmonary ischemia studied with the isolated murine lung or flow adapted pulmonary microvascular endothelial cells in vitro results in endothelial generation of reactive oxygen species (ROS) and NO. The response requires caveolae and is initiated by endothelial cell depolarization via K_ATP channel closure followed by activation of NADPH oxidase (NOX2) and NO synthase (eNOS), signaling through MAP kinases, and endothelial cell proliferation. These physiological mediators can promote vasodilation and angiogenesis as compensation for decreased tissue perfusion.     

Insect ornithine decarboxylase (ODC) complements <Emphasis Type="Italic">SPE1</Emphasis> knock-out of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are essential for cell growth, differentiation, and proliferation. This report presents the characterization of an ODC-encoding cDNA (SlitODC) isolated from a moth species, the tobacco cutworm, Spodoptera litura (Lepidoptera); its expression in a polyamine-deficient strain of yeast, S. cerevisiae; and the recovery in polyamine levels and proliferation rate with the introduction of the insect enzyme. SlitODC encodes 448 amino acid residues, 4 amino acids longer than B. Mori ODC that has 71% identity, and has a longer C-terminus, consistent with B. mori ODC, than the reported dipteran enzymes. The null mutant yeast strain in the ODC gene, SPE1, showed remarkably depleted polyamine levels; in putrescine, spermidine, and spermine, the levels were > 7, > 1, and > 4%, respectively, of the levels in the wild-type strain. This consequently caused a significant arrest in cell proliferation of > 4% of the wild-type strain in polyaminefree media. The transformed strain, with the substituted SlitODC for the deleted endogenous ODC, grew and proliferated rapidly at even a higher rate than the wild-type strain. Furthermore, its polyamine content was significantly higher than even that in the wild-type strain as well as the spe1-null mutant, particularly with a very continuously enhanced putrescine level, reflecting no inhibition mechanism operating in the putrescine synthesis step by any corresponding insect ODC antizymes to SlitODC in this yeast system.     

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37 °C. As a function of increasing pH, hL-PYK’s affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and alanine slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to alanine is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for alanine inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response.     

Exploring the interaction between the protein kinase A catalytic subunit and caveolin-1 scaffolding domain with shotgun scanning, oligomer complementation, NMR, and docking

The techniques of phage-displayed homolog shotgun scanning, oligomer complementation, NMR secondary structure analysis, and computational docking provide a complementary suite of tools for dissecting protein-protein interactions. Focusing these tools on the interaction between the catalytic sub-unit of protein kinase A (PKAcat) and caveolin-1 scaffolding domain (CSD) reveals the first structural model for the interaction. Homolog shotgun scanning varied each CSD residue as either a wild-type or a homologous amino acid. Wild-type to homolog ratios from 116 different homologous CSD variants identified side-chain functional groups responsible for precise contacts with PKAcat. Structural analysis by NMR assigned an alpha-helical conformation to the central residues 84- 97 of CSD. The extensive mutagenesis data and NMR secondary structure information provided constraints for developing a model for the PKAcat-CSD interaction. Addition of synthetic CSD to phage-displayed CSD resulted in oligomer complementation, or enhanced binding to PKAcat. Together with previous experiments examining the interaction between CSD and endothelial nitric oxide synthase (eNOS), the results suggest a general oligomerization-dependent enhancement of binding between signal transducing enzymes and caveolin-1.