Is the C-terminal region of bradykinin the binding site of polyphenols?

Bradykinin is a bioactive hormone involved in a variety of physiological processes. In various solvents, this peptide adopts beta-turn structures. The C-terminal turn is a structural feature for the receptor affinity of agonists and antagonists while the N-terminal turn might be important for antagonistic activities. Polyphenols like dimeric proanthocyanidin B3 interact with the peptide. Thus to investigate the effects of polyphenols on bradykinin activity and structure, we studied the interaction in the structuring solvent DMSO which can be a close mimic of aqueous physiological environments like receptor-binding sites. Bradykinin alone presented a folded structure with two turns. B3 interacted with the peptide C-terminus and involved the loss of the bend structure of this region, while the N-terminus turn was maintained. Numerous studies have shown that polyphenolic molecules can act upon various biological targets, and the formation of this type of complex might be one of the possible modes of action.     

Molecular genotyping of the HLA-DQ α gene region

Restriction fragment analysis has been applied to genomic DNA extracted from human tumor cell lines. Polymorphic restriction fragments encompassing the HLA-DQ gene were observed upon digestion with Bgl II, Eco RI, and Hind III. Analysis of these polymorphic fragments (or allogenotopes) showed that for each restriction enzyme a series of three differently sized allogenotopes existed. Clusters of cosegregating allogenotopes belonging to the different allelic series defined three different allogenotypes. Each allogenotype exhibited a distinctive restriction map generated by digestion with five restriction enzymes. Comparison of these restriction maps showed that generation of the polymorphisms observed at the HLA-DQ region in these sets of cell lines is not caused by a single event. In some B- and T-lymphoma cell lines a fourth allogenotype was found. The restriction site map of genomic DNA from these cell lines suggested that the latter distribution of restriction enzyme sites was most probably generated by recombination between two of the previously observed allogenotypes at a crossover site(s) adjacent to the HLA-DQ gene.     

Cloning and characterization of the immunity region of phage ?80

Summary The immunity region of phage 80 has been localized. It codes for at least three proteins: a protein of 34 kDa which has the biological properties of the phage repressor, and two other proteins of 9 kDa and 18 kDa which are the first proteins on the rightward operon. These two proteins are negatively regulated by the 34 kDa protein at a divergent promoter site. By position analogy with phage , but not by its biological activity, the 9 kDa protein could be the cro roduct. The 18 kDa protein is able to block totally UV induction of phage 80.     

Stability of plant mRNAs depends on the length of the 3′-untranslated region

Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.     

Molecular characterization of the zerknüllt region of the Antennapedia complex of D. subobscura

We have characterized at the molecular level the zerknüllt (zen) region of the Drosophila subobscura Antennapedia complex. The sequence comparison between D. subobscura and D. melanogaster shows an irregular distribution of the conserved and diverged regions, with the homeobox and a putative activating domain completely conserved. Comparisons of the promoter sequence and pattern of expression of the gene during development suggest that the regulation of zen has been conserved during evolution. The conservation of zen expression in a subpopulation of the polar cells indicates the existence of an important role in such cells. We describe a transitory segmented pattern of expression of zen in both species, suggesting the existence of interactions with a pair rule gene. Some indirect clues indicate that the z2 gene might be absent from the D. subobscura genome. A chromosome walk initiated to reach the proboscipedia gene of D. subobscura reveals that the distance between pb and zen is at least four times the one described for D. melanogaster and for D. pseudoobscura. Finally, we present cytological evidence showing that the ANT-C is inverted in D. subobscura as compared to D. melanogaster.     

No role of the 5′ untranslated region of ornithine decarboxylase mRNA in the feedback control of the enzyme

The polyamines are ubiquitous in nature and appear to fulfil several important functions, mostly related to growth, in the cell. The first, and often rate-limiting, step in the biosynthesis of the polyamines is catalysed by ornithine decarboxylase (ODC), which is subject to a variety of control mechanisms. The polyamines exert a strong feedback regulation of the expression - as well as the degradation of the enzyme. The regulation of ODC expression appears to occur at the translational level. The ODC mRNA contains a long GC-rich 5 untranslated region (UTR), which has been demonstrated to hamper the translation of the mRNA. However, it has not yet been conclusively established whether this part of the mRNA fulfils any function in relation to the polyamine-mediated control of ODC synthesis. In the present study, we have used stable transgenic CHO cells, expressing either full-length ODC mRNA or 5 UTR-truncated ODC mRNA, to elucidate the role, if any, of the 5 UTR in the translational regulation of the enzyme by polyamines. No differences in regulatory properties were observed between the cells expressing the full-length ODC mRNA and those expressing the ODC mRNA devoid of most the 5 UTR. The cell lines down-regulated ODC (synthesis as well as activity) to the same extent upon exposure to an excess of polyamines, demonstrating that the feedback control of ODC mRNA translation occurs by a mechanism independent of the major part of the 5 UTR of the ODC mRNA.     

Functional analysis of the C-terminal region of γ-glutamyl kinase of Saccharomyces cerevisiae

γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ(1)-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.     

Study of neuronal organization of the “gastric” region in the dorsal motor nucleus of the vagus nerve

The peculiarities of neuronal electrical activity in the dorsal motor nucleus (0.5–2.0 mm rostrally to the obex) were investigated in acute experiments on cats, using the microelectrode technique, under conditions of stimulation of the gastric vagal branches. In the gastric region of the nucleus, two groups of cells responding to nerve stimulation were identified: preganglionic parasympathetic neurons antidromically activated by such stimulation, and cells excited orthodromically with the involvement of afferent fibers of the vagus nerve.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 190–196, May-June, 1993.     

Determinants of laminin polymerization revealed by the structure of the α5 chain amino-terminal region

The polymerization of laminin into a cell-associated network--a key step in basement membrane assembly--is mediated by the laminin amino-terminal (LN) domains at the tips of the three short arms of the laminin αβγ-heterotrimer. The crystal structure of a laminin α5LN-LE1-2 fragment shows that the LN domain is a β-jelly roll with several elaborate insertions that is attached like a flower head to the stalk-like laminin-type epidermal growth factor-like tandem. A surface loop that is strictly conserved in the LN domains of all α-short arms is required for stable ternary association with the β- and γ-short arms in the laminin network.     

Identification of cell adhesive sequences in the N-terminal region of the laminin α2 chain

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.     

Fine structure of the ‘Wulst’ region of the domestic fowl in organotypic cultures

Summary Parts of the Wulst region of the chick embryo brain were maintained for 39 days in vitro. Processes of adjacent glial cell form zonulae occludentes and desmosomal junctions in the uppermost stratum of the cultures. Subjacent to this layer, in the neuropil, axodendritic synapses are abundant. 10–20 m below the surface the perikarya of glial cells and neurons are found. The latter form small clusters, plasma membranes of contiguous cells being directly apposed to each other. Axosomatic synapses terminate on the perikarya. Occasionally one terminal synapses on two nerve cells simultaneously. Two types of cilia arise from basal bodies in the cytoplasm of nerve cells. Type I is a slender protrusion of about 0.5 m in diameter, extending into the neuropil. On transverse sections it displays a 9 + 0 pattern of organization of paired micro tubules proximally, and an 8 + 1 configuration more distally. The length of the cilium is approximately 7 m. Type II cilia also originate in the neuronal cytoplasm. The structure of the proximal portion is identical with that of type I cilia. Toward the tip, however, the type II cilium is characterized by a bulbous enlargement which is filled with loosely folded membranes. The fine structural details of this cilium correlate closely to the outer segments of retinal photoreceptor cells during differentiation. The possible role of a light receptor in the Wulst region of birds, controlling biological rhythms, is discussed. Acknowledgements. The authors wish to thank Mrs. H. Frenk for her expert assistance in tissue culture and electron microscopic techniques     

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.