Escherichia coli expression and processing of human interleukin-1 beta fused to signal peptides

Escherichia coli expression, processing, and secretion of human interleukin-1 beta (IL-1 beta) fused to the signal peptide of E. coli OmpA or PhoA protein were studied. With fusion to either signal sequence, high-level expression was observed and the products accumulated to about 20% of total cell protein. In the fusion to OmpA leader sequence, more than 50% of the product has the OmpA signal peptide removed precisely. The majority of the processed material is not released by osmotic shock. On the other hand, very little of the material from the fusion to PhoA has the PhoA signal peptide removed. Use of the host with a mutation in prlA or prlF, variation of temperature for cell growth, and alteration of the amino acid residues around the cleavage site do not facilitate processing of the PhoA signal peptide. These results suggest that some component in the PhoA signal peptide, interacting with the Il-1 beta sequence, is interfering with the processing of the signal peptide.     

Billingen (Lower Arenig/Lower Ordovician) acritarchs from the East European Platform and their palaeobiogeographic significance

Billingen (Lower Arenig/Lower Ordovician) sediments of the St. Petersburg region, northwest Russia and the Leba area, northern Poland of the East European Craton yield acritarch assemblages, which are largely homogenous though displaying minor compositional differences that probably reflect a gradient from inner to outer shelf environments. Comparison with coeval acritarch microflora from the Yangtze Platform, South China, shows an overall similarity between Baltoscandian and South Chinese phytoplankton. The widespread uniformity in the fossil microphytoplankton may be related to the extensive global 'evae' sea-level transgression, which characterized the Billingen time. This suggests that during the Tremadoc through early Arenig times, acritarch assemblages displayed essentially an undifferentiated cold-water and oceanic character along the whole margin of Perigondwana in the South, as well as on the South Chinese and Baltic platforms, at middle latitudes (Mediterranean oceanic Realm). Despite this overall similarity, however, some typical taxa of the high-latitude Mediterranean Province (Arbusculidium, Coryphidium and Striatotheca) occur in South China, but are absent in Baltica. This discrepancy is explained as caused by differences in climatic and physiographic conditions that prevailed at the two palaeocontinents at this time. The inferred pattern of oceanic circulation during the Lower Ordovician is consistent with the palynological evidence of a prevailing warmer climate in Baltica than in South China, although the two palaeocontinents occupied the same palaeolatitudinal position.     

Genetic characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein.

Mutants defective in the structure, biosynthesis, and assembly of murein lipoprotein have been isolated. One of these mutants has been shown to synthesize a structurally altered lipoprotein. The biochemical features of the mutant lipoprotein (lipid deficiency, dimer formation, and a reduced, bound form of lipoprotein) could be attributed to a single mutation (or closely linked mutations) located at 36.4 min of the Escherichia coli map. We propose that this mutant is altered in the structural gene for murein lipoprotein (mlpA). Biochemical studies carried out with a heterogenote, mlpA/F'mlpA+, revealed the biochemical codominance of the wild-type and mutant genes.     

Cyrtocrinids (Echinodermata,Crinoidea) from Upper Jurassic Štramberk‐type limestones in southern Poland

Abstract: A systematic account of highly diverse cyrtocrinid faunules from Upper Jurassic strata of ?tramberk type (Oxfordian–Tithonian) in southern Poland (Polish Carpathians) is presented. Fourteen taxa (Phyllocrinus malbosianus, Ph. stellaris, Ph. sp., Psalidocrinus armatus, Sclerocrinus compressus, S. polonicus sp. nov., Hemicrinus aff. kabanovi, Ancepsicrinus parvus gen. et sp. nov., Tetracrinus baumilleri sp. nov., Eugeniacrinites alexandrowiczi, E. cf. moravicus, E. sp., Eudesicrinus gluchowskii sp. nov. and Hemibrachiocrinus tithonicus sp. nov. are described and illustrated. Representatives of the genus Eudesicrinus, previously recorded only from the Lower Jurassic, are here shown to extend into the uppermost Jurassic. Other cyrtocrinids considered are common in Jurassic/Cretaceous strata across Europe. In the present faunules, isocrinid (Isocrinida), comatulid (Comatulida) and roveacrinid (Roveacrinida sensu Rasmussen, inclusive of Saccocoma) crinoids are associated.     

Tissue distribution and cellular localization of hsp56, an FK506-binding protein. Characterization using a highly specific polyclonal antibody.

Heat shock protein 56 (hsp56) has been shown to be involved in two cellular pathways, as an immunophilin for FK506 and as a component of steroid receptor complexes. To help define its role in these cellular pathways, we have developed UPJ56, a polyclonal antibody raised against hsp56 purified from Jurkat cells. In Western blot experiments, hsp56 was highly expressed in rat thymus, liver, and spleen, with low levels in lung and muscle. In immunofluorescence experiments using untreated LLC-PK1 cells, fibrillar staining was seen in the cytoplasm, suggesting a cytoskeletal localization of hsp56. The nuclei were brightly stained, except for the nucleoli. Confocal microscopy demonstrated that the staining was present in all planes of the nucleus. These results suggest that hsp56 is expressed in tissues enriched in steroid receptors and is highly expressed in tissues involved in T cell function. Furthermore, the localization of hsp56 with the cytoskeleton and throughout the nucleus is consistent with its association with steroid receptor complexes.     

Crystal structure of the catalytic subunit of Cdc25B required for G2/M phase transition of the cell cycle.

Cdc25B is a dual specificity phosphatase involved in the control of cyclin-dependent kinases and the progression of cells through the cell cycle. A series of minimal domain Cdc25B constructs maintaining catalytic activity have been expressed. The structure of a minimum domain construct binding sulfate was determined at 1.9 A resolution and a temperature of 100 K. Other forms of the same co?nstruct were determined at lower resolution and room temperature. The overall folding and structure of the domain is similar to that found for Cdc25A. An important difference between the two is that the Cdc25B domain binds oxyanions in the catalytic site while that of Cdc25A appears unable to bind oxyanions. There are also important conformational differences in the C-terminal region. In Cdc25B, both sulfate and tungstate anions are shown to bind in the catalytic site containing the signature motif (HCxxxxxR) in a conformation similar to that of other protein tyrosine phosphatases and dual specificity phosphatases, with the exception of the Cdc25A. The Cdc25B constructs, with various truncations of the C-terminal residues, are shown to have potent catalytic activity. When cut back to the site at which the Cdc25A structure begins to deviate from the Cdc25B structure, the activity is considerably less. There is a pocket extending from the catalytic site to an anion-binding site containing a chloride about 14 A away. The catalytic cysteine residue, Cys473, can be oxidized to form a disulfide linkage to Cys426. A readily modifiable cysteine residue, Cys484, resides in another pocket that binds a sulfate but not in the signature motif conformation. This region of the structure is highly conserved between the Cdc25 molecules and could serve some unknown function.     

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.