Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function; tests for receptor-stimulated phosphatidylinositol turnover and for modulation of agonist binding by guanylylimidodiphosphate showed functional coupling to be present several days prior to the onset of synapse formation. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.     

Factors affecting the dormancy and germination of bleeding heart [Lamprocapnos spectabilis (L.) Fukuhara] seeds

• Information on the optimal conditions to promote the germination of Lamprocapnos spectabilis (L.) Fukuhara seeds is limited; consequently, this study was conducted to establish the requirements to break seed dormancy and promote germination.
• The selected seeds had morphophysiological dormancy and had not begun embryo development. To study the dormancy breaking and embryo development processes, seeds were subjected to constant or changing temperature treatments during moist stratification.
• High temperature and humidity resulted in vigorous embryo growth, with the longest embryos occurring after 1 month of incubation at 20 °C. At 4 °C, the seeds required incubation period of at least 3 months to germinate. Embryo growth and germination were higher with changing high and low temperatures than under a constant temperature, and changing temperatures also considerably changed the endogenous hormone levels, embryo development and germination. Bioactive gibberellin (GA) content was higher in seeds incubated at 20 °C for 1 month, then at 4 °C for 2 months. The content of endogenous abscisic acid in seeds subjected to the same treatment decreased by 97.6% compared with that of the untreated seeds.
• Embryo growth and seed germination require changing high and low temperatures; however, exogenous GA₃ could substitute for high temperatures, as it also causes accelerated germination. In this study, the seeds of L. spectabilis were identified as an intermediate simple type, a sub‐level of morphophysiologically dormant seeds.

     

Correlation between NMR spectral parameters of nucleosides and its implication to the conformation about the glycosyl bond

Analyses of high resolution proton and carbon NMR spectra of a series of guanine nucleosides in DMSO have revealed a near linear correlation between the chemical shift of the H2, atom of the sugar moiety and the vicinal coupling constant 3JC4-H1'. This unexpected result provides evidence that the variations in the glycosyl torsion angle between nucleosides in solution are less that those which have previously been reported in crystals and it is an experimental basis for analyzing the syn and anti populations from chemical shift and coupling constant data.     

Synthesis of antibacterial pseudopeptides with less hemolytic activity from a cytotoxic peptide and their pH-dependent activity

We synthesized antibacterial pseudopeptides with less hemolytic activity by incorporation of reduced amide bond ψ[CH₂NH] into α helical antibacterial peptide with hemolytic activity. As the pK_a value of reduced amide bond is 7–8, it is protonated depending on the pH. We investigated the secondary structure, the binding affinity and the leakage activity for the vesicles, and the antibacterial activity of the peptide and its pseudopeptides at neutral and basic pH. Unlike the peptide, the pseudopeptides showed a more potent leakage activity when pH increased. The peptide exhibited a lower antibacterial activity at basic pH than at neutral pH, whereas the pseudopeptide showed the same antibacterial activity at basic and neutral pH. Overall results indicated that hydrophobicity of backbone of the pseudopeptide plays an important role in the increase of leakage activity and retention of antibacterial activity at basic pH.     

Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.     

Multiple forms of alkaline phosphatase in mouse preimplantation embryos

Mouse embryos were extracted with 0.5% Triton X-100 and subjected to cellulose acetate electrophoresis. In fertilized eggs, two forms of alkaline phosphatase (ALP), a slow-moving form and a fast-moving form, were observed. As cleavage proceeded, the fast-moving form disappeared, and the slow-moving form, the mobility of which was similar to that of the slow-moving form of the kidney, became gradually dominant up to the blastocyst stage (named 'embryonic' form). With blastulation, another fast-moving form showing a similar mobility to the lung ALP began to appear in blastocysts and showed a transient dominance in hatched blastocysts. After implantation, both the embryonic form and the fast-moving form gradually faded, and were eventually replaced by the new form, which may be named 'fetal form' in Day 7 embryos. These results clearly demonstrated that ALP activity does exist in embryos at all stages of preimplantation development. Moreover, the changes in multiple forms of ALP correlated with embryonic development may suggest that these multiple forms may have differential roles in the process of early development.