Proteolytic degradation of hemoglobin by endogenous lysosomal proteases gives rise to bioactive peptides: hemorphins

Hemorphin generation by mice peritoneal macrophages has been recently reported, nevertheless no conclusive data exist to localize clearly the macrophage proteolytic activity implicated in their generation. Because lysosomes are believed to be the main site of degradation in the endocytic pathway, we have studied their potential implication in the generation of hemorphins from hemoglobin. When this protein is submitted to purified rat liver lysosomes, an early generation of hemorphin-7-related peptides, detected by a radioimmunoassay, was observed. These peptides seemed to be relatively stable during the first hours of hydrolysis.     

Immunochemical detection of vasopressin precursors: artificial processing and quantification along the hypothalamo-hypophysial axis

A novel immunological approach to the problem of the detection and molar evaluation of vasopressin precursors was taken. First, the specificity of anti-vasopressin antibodies was studied and the hormone antigenic determinant was identified as the sequence Cys-Pro-Arg-Gly-NH2. Then this antigenic determinant, not originally shared by the precursors, was reconstituted by tryptic cleavage followed by chemical fixation of glycinamide. This treatment made quantification of precursors by radioimmunoassay possible at a fmol level in various tissues. In normal rat, precursors were found only in the supraoptic nucleus (192 pmol/mg protein), paraventricular nucleus, median eminence and posterior lobe of the hypophysis. The maturation process was followed by the decrease of the ratio of precursor to hormone from 4-5 to 0.02 along the hypothalamo-hypophysial axis. In Brattleboro rats, genetically deficient in vasopressin, no precursor could be detected over the background level; that ensures the specificity and reliability of this approach.     

Detection of dermorphin-like immunoreactivity in immune tissues

Summary Site-directed antibodies against synthetic related dermorphin peptides have previously been produced and characterized. One of them, specifically recognizing the crucial ‘opioid message’ (the N-terminal part of the molecule Tyr-D-Ala-Phe-Gly), was used in the present study in order to detect and localize endogenous dermorphin-like molecules in immune tissues. Dermorphin-like peptides were found to be present in spleen and thymus of rat and mouse. The HPLC profile of the immunoreactive material showed a major peak at a retention time of 32±1 min. Purification of immune cells by panning procedures showed that both B and T cells contained this immunoreactive material. Biochemical characterization of the dermorphin-like immunoreactivity indicated that this material is a peptide resistant to aminopeptidase hydrolysis, suggesting the presence of a putative D-amino acid residue or a residue conferring resistance to a proteolytic process.     

In situ analysis of native microbial communities in complex samples with high particulate loads

In the present study a procedure combining a cell extraction method and Fluorescence In Situ Hybridization (FISH) for molecular monitoring and quantification of bacteria in soil and aquifer samples is presented. FISH was applied to bacterial cells extracted from the matrix by density gradient centrifugation. This separation method was applied to soil and aquifer samples and produced high cell recovery of 76.5%+/-4.4 and 78.0%+/-3.2, respectively. FISH, performed on the harvested cells, permitted a perfect visualization and quantification of bacteria. This approach is therefore promising for in situ detection of indigenous bacterial communities in complex samples.     

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.     

Identification of hemorphins in a cathepsin D bovine hemoglobin hydrolysate by radioimmunoassay and photodiode array detections

Morphinomimetic peptides have been purified fromhemoglobin enzymatic hydrolysates and a significantamount of evidence has been accumulated indicatingthat the generation of these peptides (hemorphins)might occur in vivo. In order to investigatetheir putative physiological role and processing fromhemoglobin in vivo, two methods were developed:a specific radioimmunoassay and a UV spectracomparison analysis. These methods were applied to acathepsin D bovine hemoglobin hydrolysate and allowedthe detection of two hemorphin-7 peptides. Thisobservation supports the putative implication ofcathepsin D in the in vivo release ofhemorphins. Among the two methods used in this study,the immunological approach exhibits highersensitivity and represents a useful method toinvestigate the in vivo role and physiologicalprocessing of hemorphins.     

Reconstitution and characterization of the human neutrophil N-formyl peptide receptor and GTP binding proteins in phospholipid vesicles

We have developed a unilamellar phospholipid vesicle system which contains the N-formyl peptide receptor and GTP binding proteins. Several detergents were investigated but only two, octyl glucoside (35 mM) and deoxycholate (7.5 mM), were capable of extracting N-formyl peptide receptor from neutrophil membranes in a form which remained functionally active upon reconstitution into phospholipid vesicles. Extracted proteins were reconstituted into phosphatidylcholine vesicles by passage over a Sephadex G-50-80 column. The reconstituted formylpeptide receptor could bind [3H]FMLP (3H-labeled fMet-Leu-Phe) and [125I]FMLPL-SASD (125I-labeled N-formylmethionylleucylphenylalanyl-N epsilon-(2-(p-azidosalicylamido)ethyl- 1,3'-dithiopropionyl)lysine) while the endogenous G protein could bind [35S]GTP gamma S. Furthermore, the functional interaction of the two proteins was preserved. Addition of the nonhydrolyzable guanine nucleotide, GTP gamma S, shifted the N-formyl peptide receptor from a high- to a low-affinity binding state for ligand. The development of this in vitro reconstitution system should provide a basis to study the mechanism of interaction of the N-formyl peptide receptor and the G protein.     

Chromatographic analysis of the enkephalin immunoreactivity in the nucleus locus coeruleus of the cat after local colchicine administration

Cell bodies immunoreactive for methionine- and leucine-enkephalin are found in the area of the locus coeruleus (dorsolateral pons) of the cat after injection of colchicine in the ascending projections of the nucleus. Using radioimmunoassay procedures, it is shown that colchicine induces a significant increase in methionine- and leucine-enkephalin-immunoreactive material in this area of the brain. High pressure liquid chromatography analysis demonstrated that the immunoreactive materials were authentic methionine- and leucine-enkephalin. The methionine- and leucine-enkephalin patterns were identical in the colchicine injected and non-injected sides of the dorsolateral pons. It is suggested that, in this area of the brain, colchicine (i) does not significantly modify the processing of proenkephalin to form the pentapeptides methionine- and leucine-enkephalin, and (ii) does not induce the appearance of new substances reactive to the enkephalin antisera employed.     

Globular protein stability: aspects of interest in protein turnover

The conformational stability of globular proteins is remarkably low. Under physiological conditions, the native globular conformation is only from 5 to 15 kcal/mole more stable than unfolded conformations. In addition, small changes in the structure of a protein such as removing one terminal residue or cleaving a single peptide bond frequently lead to a substantial decrease in the stability. Likewise, single substitutions in the amino acid sequence can increase or decrease the stability by several kilocalories per mole. The low conformational stability of globular proteins and the sensitivity to small changes in structure suggest a possible role for conformational stability in the intracellular degradation of proteins. Several lines of evidence from in vivo studies of protein degradation are consistent with this idea.