Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus

Abstract The putative ribosome binding sites preceding 32 of Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H genes were compared. A highly conserved consensus sequence for the ribosome binding sites of LL-H genes was inferred, GAAAGGAG. This study included the characterization of the last nucleotides of the 3'-end of the 16S rRNA molecule from L. delbrueckii subsp. lactis and its comparison to the ribosome binding site consensus sequence.     

The ubiquitous nature of the progesterone receptor binding factor-1 (RBF-1) in avian tissues

The avian oviduct receptor binding factor-1 (RBF-1) is a 10 kDa nuclear matrix protein that was originally identified through its ability to effect high affinity interaction of activated progesterone receptor (PR) with chromatin. In the present study, the RBF-1 is shown to not be restricted to reproductive tissues (e.g., oviduct) but present in all avian tissues examined by Western blot analysis with a monoclonal antibody prepared against purified RBF-1. The heart and pancreas had the highest and lowest RBF-1 levels, respectively; the concentration ranging by ～ 50-fold in these tissues. The 10 kDa size of the RBF-1 detected in all tissues suggests no significant tissue-specific differences in the protein. This was consistent with the finding that purified hepatic and oviductal RBF-1 have identical amino-terminal sequence. Using a recently isolated cDNA to RBF-1, the levels of RBF-1 mRNA were found to correlate well with the ubiquitous presence of the protein as well as tissue-specific differences in concentration. The presence of RBF-1 in non-progesterone responsive tissues suggests the possibility that RBF-1 may not be specifically involved in PR-DNA interactions but may play a more diverse role, possibly involving other steroid receptors such as the glucocorticoid receptor. © 1994 Wiley-Liss, Inc.     

Studies on the Topography of the Catalytic Site of Acetylcholinesterase Using Polyclonal and Monoclonal Antibodies

Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross-reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggest that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket-like conformation in the native enzyme.     

In Vivo Binding to Peripheral Benzodiazepine Binding Sites in Lesioned Rat Brain: Comparison Between [3H]PK11195 and [18F]PK14105 as Markers for Neuronal Damage

Peripheral-type benzodiazepine binding sites are not normally present in most cerebral tissues, but following neuronal damage, the cells involved in the ensuing gliosis show a marked expression of these sites. In a unilateral excitotoxic striatal lesion in the rat, we sought to determine whether the isoquinoline derivatives PK11195 and PK14105 bind to these sites in vivo and whether demonstration of these sites offers the potential of indirectly localising areas of neuronal damage. Binding was studied at several intervals after coinjection of [3H]PK11195 and [18F]PK14105 to determine the time courses of specific binding. Both compounds were rapidly extracted into all cerebral tissues, but in the absence of binding sites in nonlesioned tissues, this was followed by a rapid clearance of radioactivity. In lesioned areas, both [3H]PK11195 and [18F]PK14105 accumulated over the first 5 min followed by a much slower clearance of radioactivity, resulting in a "specific signal." [3H]PK11195 binding peaked at 20-30 min postinjection, with radioactivity in the lesioned striatum being three times greater than in its contralateral homologue. The specific signal was present for at least 60 min. The maximal [18 F]PK14105-specific signal was of similar magnitude but peaked earlier and was retained for only 45 min. Specific signals with both ligands were also detected in regions remote from the primary lesion site, e.g., in the hippocampus and substantia nigra. Predosing animals with a large dose of PK11195 (3 mg/kg), sufficient to saturate peripheral-type benzodiazepine binding sites, abolished in vivo binding of both [3H]PK11195 and [18F]PK14105 to both primary- and remote-lesioned tissues. The specific signal with both ligands could be of sufficient magnitude and duration to make tomographic studies in humans feasible.     

Methyllycaconitine: a selective probe for neuronal α-bungarotoxin binding sites

The ability of methyllycaconitine (MLA) to inhibit the binding of [¹²⁵I]α-bungarotoxin to rat brain membranes, frog and human muscle extracts and the human muscle cell line TE671 has been measured. MLA showed a markedly higher affinity for the rat brain site (K_i 1.4 × 10⁻⁹ M) than for the muscle receptors (K_i; 10⁻⁵-10⁻⁶ M). Structure modelling techniques were used to fit the structure of MLA to a nicotinic pharmacophore model. MLA is the first low molecular weight ligand to be shown to discriminate between muscle nicotinic receptors and their α-bungarotoxinbinding counterpart in the brain, and as such may be a useful structural probe for pursuing the structural and functional properties of the neuronal protein.     

Anti-predator strategies in oviposition site selection of Pyrrhosoma nymphula (Zygoptera: Odonata)

Summary Selection of sites for oviposition when the risk of predation by green frogs was variable studied in tandems of the damselfly Pyrrhosoma nymphula. In the absence of predators the number of tandems landing increased with the size of the oviposition site and with the number of pairs already present. Pairs approaching an oviposition site landed promptly and preferred locations near other ovipositing pairs. Pairs which had landed near others left the landing site and flew to another water plant after a shorter period than those landing alone. They stayed in groups for only 16.5% of the total duration of stay at the oviposition site. With predators present fewer tandems landed. There was no relationship between the landing site of solitary tandems and the locations of the perching frogs. Pairs landing in groups were disturbed by frogs after a shorter period than pairs landing alone. Damselfly aggregation did not affect the predation success of the frogs, but the predation risk to individual tandem pairs was reduced.     

Does cobalt really bind tightly to hemocyanin active sites?

The analysis of Co(II)-apoHc complexes of two arthropodan species (freshwater crayfish): Orconectes limosus and Astacus astacus enabled to reach some conclusions about possible cobalt binding sites in the hemocyanin molecules. The occurrence of binding sites for Co(II) at sites other than the active center has been demonstrated. We excluded the possibility of strong binding of EDTA-non-removable cobalt ions in the binding sites occupied by copper. There were no differences between apoHc and the Co(II)apoHc complex in terms of the amount of bound Cu(I) ions and the kinetics of Cu(I) ion reconstitution.Abbreviations He hemocyanin - apoHc apohemocyanin - oxyHc oxyhemocyanin - Co-Hc hemocyanin complex with cobalt ions Offprint requests to: E. Serafln     

Studies on inactivation of anion transport in human red blood cell membrane by reversibly and irreversibly acting arginine-specific reagents

Summary A chromophoric derivative of phenylglyoxal, 4-hydroxy-3-nitrophenylglyoxal (HNPG), known to be highly selective for modification of arginine residues in aqueous solution is found to be a potent inhibitor of anion transport across the red cell membrane. In contrast to the action of all other arginine-specific reagents used under the experimental conditions in this laboratory, the action of HNPG on sulfate transport is completely reversible. Hence, a kinetic analysis of its inhibitory effect on SO ₄ ^2– self-exchange could be performed. The effect of increasing chloride concentration on the inhibitory potency of HNPG is consistent with the concept that Cl^– and HNPG compete for the same site on the anion transporter. The IC₅₀ value for the inhibition of SO ₄ ^2– exchange with HNPG is about 0.13mm at pH 8.0 and 0.36mm at pH 7.4, and the Hill coefficient for the interaction between the transporter and the inhibitor is near one at both pH's. HNPG is able to protect the transport system against inhibition with the (under our experimental conditions) irreversibly acting arginine specific reagent, phenylglyoxal. Partial inactivation of the transport system with phenylglyoxal lowers the maximal rates of SO ₄ ^2– and chloride exchange but does not modify the apparentK _s for the substrate anions. Reversibly acting anion transport inhibitors known to interact with the DIDS binding site like salicylate, tetrathionate, APMB, DNDS, and flufenamate are able to protect the transport system against phenylglyoxalation. Other inhibitors like phloretin and phlorizin have no effect.     

Heterogeneity of γ-Aminobutyric Acid/Benzodiazepine/β-Carboline Receptor Complex in Rat Spinal Cord

The properties of muscimol, beta-carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high-affinity [3H]muscimol and [3H]6,7-dimethoxy-4-ethyl-beta-carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or [3H]flumazenil (Ro 15-1788; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1-4]benzodiazepine-3-carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is approximately 20-25% of the number of FLU or flumazenil binding sites. Moreover, in spinal cord membranes, BC recognition site ligands displace [3H]-flumazenil bound to those sites, with low affinity and a Hill slope significantly less than 1; the potency of the different BCs in displacing [3H]flumazenil is 20-50-fold lower in the spinal cord than in the cerebellum. [3H]Flumazenil is not displaced from spinal cord membranes by the peripheral BZD ligand Ro 5-4864 (4'-chlorodiazepam), whereas it is displaced with low affinity and a Hill slope of less than 1 (nH = 0.4) by CL 218,872 (3-methyl-6-(3-trifluoromethylphenyl)-1,2,4-triazolol[4,3-b] pyridazine). These data suggest that a large number of BZD binding sites in spinal cord (approximately 80%) are of the central-type, BZD2 subclass, whereas the BZD binding sites in cerebellum are predominantly of the central-type, BZD1 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and Characterization of the Serotonin 5-HT1D Receptor in Rat and Human Brain

In the presence of 1 microM ( +/- )-pindolol [to block 5-hydroxytryptamine (5-HT, serotonin) 5-HT 1A and 5-HT 1B receptors] and 100 nM mesulergine (to block 5-HT 1C receptors), 2.0 nM [3H]5-HT binding to rat cortical homogenates is specific, saturable, and reversible. Scatchard analysis of [3H]5-HT binding, in the presence of 1 microM ( +/- )-pindolol and 100 nM mesulergine, produced a KD of 3.2 nM and Bmax of 43 fmol/mg protein. Distribution studies show this site to be present in most rat brain regions. This site is also detectable in human caudate. The pharmacological profile of this site is distinct from the previously identified 5-HT receptor subtypes. Compounds with high affinity for 5-HT 1A (8-hydroxydipropylaminotetralin), 5-HT 1B (trifluoromethylphenylpiperazine), 5-HT 1C (mesulergine), 5-HT 2 (4-bromo-2,5-dimethoxyphenylisopropylamine), and 5-HT3 (ICS 205-930) receptors have low affinity for this site. These data suggest the presence of an additional, previously unidentified, 5-HT binding site in rat and human brain tissue. This putative novel 5-HT receptor has a similar pharmacology to the "5-HT 1D" site detected in bovine brain by Heuring and Peroutka.