Biomedica biochimica acta最新文献

Using isolated hepatocytes as a model system we have investigated whether the cyclic nucleotides cAMP and cGMP are involved in the regulation of the autophagic process. The dibutyryl-cyclic nucleotide analogues db-cAMP and db-cGMP both inhibited autophagic sequestration, suggesting that cAMP and cGMP may be of significance for this step. The adenylate cyclase stimulator deacetyl-forskolin both raised the level of intracellular cAMP and reduced sequestration markedly. In contrast, the guanylate cyclase stimulating agent atriopeptin did not affect sequestration although, it effectively elevated, the level of cGMP. Several inhibitors of cyclic nucleotide phosphodiesterases strongly suppressed autophagy and elevated the level of both cAMP and cGMP. However, one inhibitor, milrinone, raised the cAMP level 3-4 x while having no significant effect on cGMP. These results suggest that cAMP may be involved in the control of hepatic autophagy, whereas the role of cGMP, if any, remains unclear.

Calcium-phosphate induced fusion of human erythrocytes was investigated using an assay based on the relief of the fluorescence selfquenching of a fluorescent amphiphile incorporated into ghost membranes as it occurs when labeled membranes are fused with unlabeled membranes. Measuring ghost fusion up to Ca(2+)-concentrations of 4 mM in the presence of 10 mM phosphate, both an acceleration of the fusion process and an enhancement of the fusion extent with increasing amounts of calcium were observed. Fusion takes place even in the case when calcium-phosphate complexes were formed in the suspension medium prior to the addition of ghosts. These results are in contrast to previous observations, obtained by an internal aqueous content mixing assay, where fusion of ghosts was inhibited at Ca(2+)-concentrations greater than 1.75 mM, and preformed calcium-phosphate complexes were not able to induce fusion. The results point to the necessity of utilizing content mixing assays in conjunction with the lipid dilution assay to get a more detailed insight in membrane fusion mechanism(s).

A class of cytosolic proteins has been identified that are degraded faster (have shorter half-lives) in human diploid fibroblasts deprived of serum. In RNase A, a model protein used for these studies, a pentapeptide comprising amino acids 7-11, Lys-Phe-Glu-Arg-Gln or KFERQ, is responsible for its enhanced degradation. The cytosolic proteins that are degraded faster during serum deprivation are recognized by an antiKFERQ antibody and, therefore, probably contain variations of the KFERQ motif. These cytosolic proteins are degraded in lysosomes. Transport into lysosomes in vitro is stimulated by ATP and the heat shock cognate protein of 73 kDa (hsc73).

The mRNA for the lysosomal proteinases cathepsins B, D, H, L, and S are broadly distributed in normal rodent tissues. Although total cathepsin mRNA levels generally parallel the protein catabolic activity of the tissues, the expressions of the individual enzymes do not appear to be linked. Thus, the relative proportions of the individual messages are found to vary from tissue to tissue. Further evidence for the independent regulation of lysosomal proteinase expression is derived from observations of selective increases in mRNA levels for individual proteinases in rodent tumors. Only cathepsin B mRNA is elevated in a highly metastatic murine B16a melanoma and in a Walker-256 rat carcinosarcoma, while Moloney murine sarcoma virus-transformed fibroblasts express increased mRNA for cathepsins B, D, and L and normal levels for H and S. To address the regulation of cathepsin B expression, the mouse cathepsin B gene and its 5'-upstream region were cloned. The gene has 10 exons and 9 introns spanning about 20 kilobases. The 5'-upstream region and exon 1 are GC-rich with several potential Sp1 binding sites. TATA and CAAT motifs adjacent to the transcription start site are not evident. These properties are characteristic of mammalian "housekeeping" genes. B16 melanoma cells contain three cathepsin B transcripts of 2.2, 4.0 and 5.0 kilobases. The two larger messages, which were not found in normal tissues, contain unusually long 3'-untranslated regions resulting from the alternative cleavage and polyadenylation of the 3' end of the cathepsin B pre-mRNA in B16 melanomas. As all three messages encoded normal preprocathepsin B, cathepsin B secretion by melanoma cells is probably due to posttranslational mechanisms and not to alternative splicing or gene mutation.

The theory of steady-state flux control was applied to characterize the regulation of beta-oxidation flux in uncoupled rat liver mitochondria oxidizing palmitoylcarnitine in the presence of rotenone, malonate and the beta-hydroxybutyrate/acetoacetate redox buffer. By titrations with inhibitors such as antimycin, myxothiazol, azide and 4-pentenoic acid, the flux control coefficients of the b-c1 complex, cytochrome c oxidase and thiolase, were determined experimentally. The flux control coefficients of carnitine palmitoyltransferase II, ETF:CoQ oxidoreductase and beta-hydroxybutyrate dehydrogenase were determined from elasticity coefficients obtained by measuring the flux dependencies of acyl-CoA and acetyl-CoA+CoASH concentrations, the electron transfer flavoprotein redox state, the CoQ redox state and the NAD redox state. It was found that at low flux rates the flux control was distributed mainly between acyl-CoA dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase (Ci = 0.89). At maximum flux rates, carnitine palmitoyltransferase II (Ci = 0.35) and thiolase (Ci = 0.13) contribute additionally to the flux control. Thus, the phenomena of regulation of mitochondrial beta-oxidation can be described as multistep control.

A series of hydropolyborate salts were observed to possess hypolipidemic activity in rodents. Tetramethylammonium octahydrotriborate, tetramethylammonium hexahydrohexaborate, tetramethylammonium dodecahydrododecaborate and triethyl-ammonium dodecahydrododecaborate proved to be very effective in lowering serum cholesterol and triglyceride levels in CF1 mice, i.p. and Sprague Dawley rats, orally. Tissue lipids were reduced by these agents e.g. liver cholesterol, and triglyceride and small intestine mucosa cholesterol levels. [3H] Cholesterol distribution studies confirm that steroid levels are lower in most major tissues. The rat serum lipoprotein lipid content was altered by drug treatment, with cholesterol and triglyceride being reduced in the VLDL and cholesterol being reduced in the LDL. HDL cholesterol levels were elevated by drug treatment. The fecal lipids were increased with select derivatives. The enzyme activities involved in de novo synthesis of hepatic lipids were affected by the hydropolyborate salts including cytoplasmic ATP-dependent citrate lyase, acetyl CoA synthetase, acyl CoA cholesterol acyl transferase, sn-glycerol-3-phosphate acyl transferase and phosphatidylate phosphohydrolase.

The (K15R M52E) aprotinin is a recombinant molecule with a broader inhibition spectrum against serine proteinases and a higher affinity towards certain proteinases as compared to native aprotinin. This aprotinin variant was produced in E. coli, isolated and purified to homogeneity. The inhibitor was further tested for its effectiveness to reduce human immunodeficiency virus type 1 (HIV-1) replication. Virus growth was followed by an ELISA which detects the amount of virus core protein p24. At a concentration of 50 microM, the recombinant (K15R M52E) aprotinin clearly reduced HIV-1 replication in H9 cells.

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.

The effect of dextran sulfate on the fusion of Sendai virus and erythrocyte ghosts was studied as a function of dextran sulfate concentration and pH of cell suspension solutions. In order to examine the interaction of dextran sulfate with Sendai virion and erythrocyte ghost surfaces, the turbidity of cell suspensions was also measured with respect to the same parameters as above. It was found that dextran sulfate inhibited the fusion of Sendai virus with erythrocyte ghosts. The lower the pH of the suspension solution was, the more effective was dextran sulfate in suppressing virion erythrocyte ghost fusion. Dextran sulfate of a higher molecular weight was slightly more effective in suppressing fusion than that of a lower molecular weight. From turbidity measurements of Sendai virus and erythrocyte ghost suspensions, it is likely that dextran sulfate interacts preferentially with Sendai virion surfaces and inhibits interaction between Sendai virions and erythrocyte ghosts.

Specific labelling of glycogen phosphorylase with the precursor of the cofactor has allowed definition of the turnover parameters of the enzyme in the pectoralis of rapidly growing broiler chickens, and slowly growing layer chickens. Selection for rapid growth rate in broilers has resulted in a lower rate of turnover of phosphorylase, but the difference between rates of synthesis and degradation is maintained, which contrasts markedly with the age-dependent coalescence of the two values in layer chickens.