Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation

A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 Ile29 Leu89) chicken cystatin was cloned and expressed in E. coli. The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8. Expression as fusion protein was performed in a temperature-inducible E. coli system. The expression product was synthesized as 20% of total E. coli protein. The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N-terminal sequence confirmed up to position 37. The properties of the purified material correspond to those of natural chicken cystatin. The recombinant cystatin variant binds anti-chicken cystatin IgG, is inhibitorily active and displays Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin.     

Structural and membrane modifying porperties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in black lipid films.

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete condustance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger alpha-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of "inactivation".     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part B. Pore formation in black lipid films

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete conductance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation tion starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger α-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of “inactivation”.     

Design,synthesis, biological evaluation and QSAR analysis of novel N-substituted benzimidazole derived carboxamides

As a result of our previous research focussed on benzimidazoles, herein we present design, synthesis, QSAR analysis and biological activity of novel N-substituted benzimidazole derived carboxamides. Carboxamides were designed to study the influence of the number of methoxy groups, the type of the substituent placed at the benzimidazole core on biological activity. Pronounced antioxidative activity displayed unsubstituted 28 (IC₅₀ ≈ 3.78 mM, 538.81 mmolFe²⁺/mmolC) and dimethoxy substituted derivative 34 (IC₅₀ ≈ 5.68 mM, 618.10 mmolFe²⁺/mmolC). Trimethoxy substituted 43 and unsubstituted compound 40 with isobutyl side chain at N atom showed strong activity against HCT116 (IC₅₀ ≈ 0.6 µM, both) and H 460 cells (IC₅₀ ≈ 2.5 µM; 0.4 µM), being less cytotoxic towards non-tumour cell. Antioxidative activity in cell generally confirmed relatively modest antioxidant capacity obtained in DPPH/FRAP assays of derivatives 34 and 40. The 3D-QSAR models were generated to explore molecular properties that have the highest influence on antioxidative activity.     

Iron‐regulated surface determinant B (IsdB) promotes Staphylococcus aureus adherence to and internalization by non‐phagocytic human cells

Staphylococcus aureus is a human pathogen that causes invasive and recurring infections. The ability to internalize into and persist within host cells is thought to contribute to infection. Here we report a novel role for the well‐characterized iron‐regulated surface determinant B (IsdB) protein which we have shown can promote adhesion of 293T, HeLa cells and platelets to immobilized bacteria independently of its ability to bind haemoglobin. IsdB bound to the active form of the platelet integrin α_IIbβ₃, both on platelets and when the integrin was expressed ectopically in CHO cells. IsdB also promoted bacterial invasion into human cells. This was clearly demonstrated with bacteria lacking fibronectin‐binding proteins (FnBPs), which are known to promote invasion in the presence of fibronectin. However, IsdB also contributed significantly to invasion by cells expressing FnBPs in the presence of serum. Thus IsdB appears to be able to interact with the broader family of integrins that bind ligands with the RGD motif and to act as a back up mechanism to promote interactions with mammalian cells.     

Ground beetle assemblages and distribution of functional traits in olive orchards and vineyards depend on the agricultural management practice

BioControl - Biodiversity in the Mediterranean is undergoing a decline resulting in reduced ecosystem service provisions. Here we analysed the functional diversity of ground beetles in...     

Antimetabolic activity of L-ascorbic acid in human and animal tumors

1. The effect of high concentrations of L-ascorbic acid on the growth of some human and animal transformed and non-transformed cell lines has been investigated. Directly implemented into culture of transformed cell lines it decreased [3H]thymidine, [3H]uridine and [3H]leucine incorporation into cells. Vitamin C inhibited DNA synthesis by transformed cells 3-4 times more efficiently than by normal cells. 2. In vivo treatment of athymic nude mice bearing human mammary carcinoma with 500 mg/kg L-ascorbic acid for the first 15 days markedly inhibited the growth of tumor cells. 3. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in mammary carcinoma cells treated with vitamin C. DNA-DNA and DNA-protein cross links in cells treated with L-ascorbic acid were revealed by the proteinase K assay. Removal of vitamin C caused an immediate onset of spontaneous repair of single or double stranded DNA breaks. If, however, vitamin was reintroduced into cell culture, this spontaneous repair was reversed. 4. Our results indicate an antimetabolic activity of L-ascorbic acid in human and animal transformed cells, probably due to lethal damages in DNA.     

Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.