Biomedica biochimica acta最新文献

The brush border membrane of mice and rats contains a phosphoramidon-insensitive metalloproteinase, meprin (neutral endopeptidase-2; NEP-2). The role of meprin is unknown, but we have shown that urine from these species contains insulin B chain degrading activity that is due to a phosphoramidon-insensitive metalloendopeptidase. By enzymic and immunological criteria, it is likely that this activity is due to meprin, and introduces the possibility that this enzyme may have a role in urinary function.

Through a study on thermal degradation of fish jelly products, the existence of a group of latent trypsin-like serine proteinases was demonstrated in fish muscle. These proteinases share common properties in existing as latent forms (being activated by heating around neutral pH in the presence of NaCl), having trypsin-like serine proteinase properties and showing strong myosin heavy chain degrading activity. This group of proteinases could be classified into four subtypes according to the intracellular localization (sarcoplasmic and myofibril-associated types) and the optimum temperature range (50 and 60 degrees C types).

Tumor cell invasion and metastasis is a multifactorial process, which at each step may require the action of proteolytic enzymes such as collagenases, cathepsins, plasmin, or plasminogen activators. An enzymatically inactive proenzyme form of the urokinase-type plasminogen activator (pro-uPA) is secreted by tumor cells which may be converted to an enzymatically active two-chain uPA-molecule (HMW-uPA) by plasmin-like enzymes. Action of proteases on pro-uPA may generate the enzymatically active or inactive high-molecular-weight form of uPA (HMW-uPA). Some proteases (plasmin, cathepsin B and L, kallikrein, trypsin or thermolysin) activate pro-uPA by cleaving the peptide bond Lys158 and IIe159. Other proteases (elastase, thrombin) cleave pro-uPA at different positions to yield enzymatically inactive HMW-uPA. HMW-uPA may be split into the enzymatically active LMW-uPA and the enzymatically inactive ATF (amino terminal fragment). ATF may be cleaved between peptide sequence 20 and 40 within the receptor binding domain of uPA (GFD). Such impaired ATF does not bind to uPA-receptors. Action of the bacterial endoproteinase Asp-N from Pseudomonas fragi mutant on pro-uPA or HMW-uPA, however, generates intact ATF which efficiently competes for binding of HMW-uPA or pro-uPA to receptors on tumor cells. High uPA-antigen content (pro-uPA, HMW-uPA, or LMW-uPA) in breast cancer tissue (not in plasma) indicates an elevated risk for the patient of recurrences and shorter overall survival. Thus pro-uPA/uPA-antigen content in breast cancer tissue serves as an independent prognostic parameter for the outcome of the disease. Cathepsin D is also an independent prognostic factor for recurrences and overall survival. High content of cathepsin D in breast cancer tumors is, however, not correlated with elevated levels of pro-uPA/uPA indicating that synthesis and release of cathepsin D and pro-uPA/uPA are independent events.

According to the difference of molecular masses estimated by SDS-polyacrylamide gel electrophoresis, calpastatins are classified into two types, i.e. muscle type (110 kDa) and erythrocyte type (70 kDa). Muscle type calpastatin contains four internally repetitive sequences (Domains 1-4) and one nonhomologous sequence on the amino-terminal side (Domain L), whereas erythrocyte type lacks Domains L and 1. By immuno-blot analysis, chicken erythrocytes, nucleated cells, were found to contain muscle type calpastatin. In avian erythrocytes, diminution of the calpastatin molecule as in mammalian erythrocytes was not observed.

Vascular endothelial cells undergo morphological and functional changes at sites of cell-mediated immune responses which may serve to promote the pathogenesis of inflammation. These changes, described as "endothelial cell activation" can be invoked by a variety of cytokines which include interleukin I (IL-1), tumor necrosis factor (TNF), and lipopolysaccharide (LPS). We report here on the regulation of the plasminogen activator (PA) proteolytic system by human recombinant TNF alpha in short term cultures (less than 4 passages) of human umbilical vein endothelial cells (HUVECs). TNF alpha treatment of HUVECs enhanced the production of 55 kDa urokinase (u) PA activity and uPA antigen by fourfold, in a concentration dependent manner (5-100 U/ml), following a 24 h treatment as determined by PA zymography and micro-ELISA assays, respectively. This response was specific for uPA since, no change in extracellular tissue type PA activity and tPA antigen levels were noted under analogous conditions. A similar 4-fold increase in the de novo synthesis of [35S]-methionine radiolabeled uPA was observed by immunoprecipitation following a 24 h TNF treatment. The induction of uPA by TNF was inhibited by actinomycin D and cycloheximide implying the necessity of RNA and protein synthesis, respectively. The effect of TNF could not be prevented by the addition of IL-1 neutralizing antibodies. Therefore, it is unlikely that TNF acts through the induction of IL-1 secretion. Time course studies using PA zymography indicate that within 8 h after TNF exposure, a 2-fold increase in uPA activity above untreated basal levels was observed. Upregulation of extracellular uPA production in HUVECs following TNF treatment suggests yet a new aspect of cellular and interstitial PA regulation in endothelium during inflammation and angiogenesis.

Until recently, the "substrate-like" "canonical" inhibition by the "small" serine proteinase inhibitors, and the product-like inhibition by the carboxypeptidase inhibitor, provided the only models for protein inhibitor-proteinase interactions. The recently published structures of cystatin/stefin-papain complexes and of hirudin-thrombin complexes reveal novel modes of interactions of only partial substrate-like character. Despite considerable progress in understanding the native-cleaved transition of the serpins, the mechanisms of their interaction with their cognate serine proteinases is still a matter of conjecture.

The effect of four human serine proteinases on the human cysteine proteinase inhibitor, cystatin C, has been studied in vitro. Neutrophil elastase in catalytic amounts was observed to rapidly cleave cystatin C at neutral pH, thereby giving rise to a modified form of the inhibitor lacking the N-terminal Ser1-Val10 decapeptide. The two other leukocyte serine proteinases, cathepsin G and neutrophil proteinase 4, did not catalytically hydrolyse cystatin C bonds. Neither had the seminal plasma serine proteinase, prostate-specific antigen, any effect on cystatin C. The physiological implications of neutrophil elastase catalysed modification of cystatin C are discussed, and recent findings indicating that this reaction also occurs in vivo are reviewed.

Recent developments in the enzymatic manipulation of protecting groups are reviewed. The application of enzymes opens new alternatives in the methodics of peptide synthesis. The following topics are dealt with: 1. New combinations of protecting groups 2. Simultaneous deprotections 3. Enzymatic regiospecificity versus chemical selectivity 4. Racemization control and enantioselective deprotection 5. N- and O-glycosylated amino acid and peptide derivatives.

Four different supports: polyacrylamide (I), polyethylene glycol-polystyrene MP-PEG (II), Kel-F-g-styrene (III) and controlled pore glass CPG (IV-VI) were tested in order to determine the most suitable carrier for both chemical and enzymatic methods. As a model of enzymatic reaction for comparative study of supports, the hydrolysis of peptide methyl esters bound to insoluble polymer was examined. Peptide segment couplings by means of papain were attempted on the CPG-support which was found to be the most suitable for performing enzymatic reactions. The racemization-free assemblies of partially protected peptides on this support were achieved in moderate yields (31-54%).

Groups of each 8 male Long-Evans rats were treated with 1 or 10 micrograms/kg LHRH intraperitoneally and compared with control rats which received the vehicle fluid (NaCl-solution). Ambulatory activity in an open field (OF), entrance to central fields and the mobility index were significantly decreased by both doses. Correspondingly, wheel running and movement velocity were significantly decreased. A further group of 8 rats with chronically implanted electrodes which was habituated to sessions showed an increase of slow-wave sleep and a significant reduction of waking and active states with maximal expression 45 min after ip application of 10 micrograms/kg LHRH. Paradoxical sleep was slightly reduced. Sleep-wakefulness cyclograms showed increase of a sleep phase duration to 175% and of single slow wave sleep phase duration to 140% after LHRH. Phases of drowsiness were also prolonged.