Biological chemistry Hoppe-Seyler最新文献

Ochratoxin A is a mycotoxin produced by Aspergillus ochraceus and is a natural contaminant of mouldy food. We examined the neuroactive potential of ochratoxin A by measuring the changes in the activities of several membrane bound, cytoplasmic and lysosomal enzymes in the brain of adult female rats, following subchronic application of ochratoxin A. The activities of both soluble and membrane bound fractions of ecto-5'nucleotidase, ecto-Ca2+/Mg2+ATPase, alanine aminopeptidase, gamma-glutamyl transferase, as well as activities of lactate dehydrogenase and of N-acetyl-beta-D-glucosaminidase were followed. Biochemical effects were examined in cerebral cortex, cerebellum and hippocampus. The results obtained showed physiologically significant alterations in the activity of enzymes tested. The changes were found to be time-dependent and regionally selective. Compared to controls, statistically significant increases in gamma-glutamyl transferase were observed in all three brain regions, while in the case of alanine aminopeptidase activities differed with regard to region, the highest increase being observed in hippocampus. Ecto-Ca2+/Mg2+ATPase and ecto-5'nucleotidase showed distinct changes lasting for 20 days of treatment, while increase in the activities of N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase were visible only at the beginning of the treatment. By the end of the trial the activities of almost all enzymes returned back to normal values.

One of the most surprising observations made in plant science in recent years is the inactivation of transgenes triggered by interactions between DNA repeats. In plants, we can differentiate between transcriptional silencing, most likely reflecting a regulation at the DNA level, and post-transcriptional silencing that affects steady state RNA levels. In the filamentous fungi Ascobolus immersus and Neurospora crassa, we find two premeiotic silencing processes that are also based on the interaction of repeated sequences. A common feature of transcriptional silencing in plants and premeiotic gene inactivation in filamentous fungi is that the repeated sequences undergo cytosine methylation. DNA methylation, which is either the cause or the consequence of gene silencing, can be associated with changes in chromatin structure. These structural changes are reminiscent of homology-based silencing mechanisms in Drosophila, an organism that lacks DNA methylation. Repeat-induced silencing may therefore reflect the activity of an endogenous mechanism, present in some species, which screens for homology and has significant implications for the organization and evolution of the genome.

The Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus and a causative agent of the Acquired Immuno Deficiency Syndrome (AIDS). Retroviruses are distinct from other viruses in their ability to encode an enzyme called reverse transcriptase (RT). The RT is the enzyme mainly involved in replication. It performs RNA- as well as DNA-dependent DNA synthesis in order to convert the single-stranded viral RNA genome into double-stranded DNA. The double-stranded DNA is stably integrated into the host cell genome and is used as a template for the production of a new viral generation. The HIV-1 RT is partially encoded by the POL open reading frame of the HIV-1 genome and consists of two subunits of 66 kDa (p66) and 51 kDa (p51). The p66 polypeptide encodes the reverse transcriptase and the RNase H domain. Half of the p66 molecules are further processed to generate the p51 protein with an identical N-terminus, but lacking the C-terminus which encodes the RNase H domain. In vivo both polypeptides are found in equimolar amounts thus forming a heterodimer. This dimerization is critical for the enzymatic activity. In this review we summarize (i) the replication cycle of HIV-1, (ii) the enzymatic properties of HIV-1 RT and (iii) the structure-function relationship of the HIV-1 RT in view of the known three dimensional structure.

From a murine B-cell cDNA-library we have cloned a cDNA encoding the murine B-cell specific coactivator mBob1. The protein is the murine homologue to the recently described human coactivator Bob1 (hBob1), also referred to as OBF-1 or OCA-B. We have also characterized the genomic mBob1 clone. Analysis of its intron-exon structure has allowed identification of a C-terminal splice variant. mBob1 is B-cell restricted, and is found in all B-cell lines representing different stages of B-cell differentiation. mBob1 interacts with the octamer transcription factors Oct-1 and Oct-2 and stimulates transcription mediated by these factors.

Insulin from a monotreme, the platypus (Ornithorhynchus anatinus), was isolated and the amino acid sequence determined. It differs from pig insulin at eleven amino acid sites, mainly on the surface of the monomer. Substitutions relative to pig insulin occur in the monomer-monomer interface, the dimer-dimer interface and the receptor binding region. The residues A5 Glu, A8 Lys and A13 Met have not been reported before in any insulin. Multiple sequence comparison studies reveal a relatively close relationship with the nearest group of relatives to the platypus, the mammals. The relationship of the platypus sequence to reptilian insulin sequences (and amphibian and avian insulin sequences in this case) is sufficiently close to support the observation that platypus has retained some ancient reptilian characteristics over the course of evolution. Model building the platypus insulin sequence on the structure of porcine insulin indicates that there may be some interesting differences.

Proteolytic enzymes have many physiological functions in plants, bacteria, viruses, protozoa and mammals. They play a role in processes such as food digestion, complement activation or blood coagulation. The action of proteolytic enzymes is biologically controlled by proteinase inhibitors and increasing attention is being paid to the physiological significance of these natural inhibitors in pathological processes. The reason for this growing interest is that uncontrolled proteolysis can lead to irreversible damage e.g. in chronic inflammation or tumor metastasis. This review focusses on the possible role of the cystatins, natural and specific inhibitors of the cysteine proteinases, in pathological processes.

Cystatin C, a low Mr cysteine proteinase inhibitor was isolated from bovine parotid glands by a procedure which includes alkaline treatment of the homogenate, affinity chromatography, gel filtration and ion exchange chromatography. The purified inhibitor has a pl of 8.0 and Mr of 14500. The identity with bovine cystatin C from colostrum was confirmed by N-terminal sequence of the inhibitor and amino acid composition. Cystatin C rapidly (kass = 5.5 x 10(7) M-1s-1) and tightly inhibits papain (Ki = 0.02 nM), whereas its interaction with bovine cathepsin B is substantially weaker (Ki = 4.4 nM). Bovine cystatin C also shows a weak antiviral effect on poliovirus infected human Hela cells.

The human glutathione transferase (GST, EC 2.5.1.18) alpha class locus comprises several genes and pseudogenes. Genomic DNA encoding several human alpha-class-related genes and pseudogenes was cloned and characterized. Three distinct but highly similar 5'-flanking regions of GST alpha genes as well as a series of 5'-deletions were investigated for promoter activity by fusion to the luciferase reporter gene. Transient transfection of these luciferase constructs into human hepatoblastoma, kidney carcinoma, nephroblastoma or bladder carcinoma cells revealed that the promoters are active and contain both positive and negative regulatory regions that behave in a cell-type specific fashion. The 150 bp proximal promoter regions of the three sequences retained the same relative activities as the full length promoters. Two of them were equally active, whereas the third one showed only 20% of the activity of the two stronger promoters. Site-directed mutagenesis indicated that a conspicuous insertion of three nucleotides (TTT) in the weak promoter is not responsible for the different activities.

In single pass perfused rat liver, rapid osmotic water shifts across the plasma membrane in response to hyperosmolar urea were followed by monitoring liver mass and transient concentrating or diluting effects on Na+ concentration in effluent perfusate. Sudden addition or removal of hyperosmolar urea (200mM, resulting in a step change of the perfusate osmolarity from 305 to 505 mosmol/l) had little effect on liver mass or Na+ activity in the effluent perfusate, suggesting that urea equilibrated at a rate similar to that of water across the liver plasma membrane. When, however, phloretin (0.2mM) was present, sudden addition (removal) of urea (200mM) induced within seconds a marked and transient decrease (increase) of both liver mass and effluent Na+ concentration, suggestive of transient osmotic water shifts out of/into the cells. Although to a lesser extent, comparable effects were induced when urea was added/removed in the presence of the phloretin-related phenol compounds 2,4,6-trihydroxyacetophenone (5mM) and 2,4,5-trihydroxybutyrophenone (5mM). Phloretin-induced inhibition of urea export from livers preloaded with [14C]urea was reversible, and no saturation of urea transport was found at concentrations up to 200mM. In contrast to [14C]urea transport, [3H]water transport across the plasma membrane was not affected by phloretin. The data indicate that urea export across the hepatocyte plasma membrane is almost as fast as water export. The urea transport mechanism is sensitive to phloretin and other phenol compounds, works with high capacity and is distinct from the water-transporting system. The regulation of this putative transport mechanism and its relevance for hepatic nitrogen metabolism remain to be established.