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.     

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.     

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”.     

In vitro compatibility of Beauveria bassiana strain ATCC 74040 with copper fungicides

Copper fungicides and mycoinsecticides based on entomopathogenic fungi Beauveria spp. are the most common pesticides used in organic crop production systems. The in vitro effects of the copper fungicides copper oxide, copper hydroxide, copper oxychloride, copper sulphate, dicopper chloride trihydroxide and tribasic copper sulphate were investigated for mycelial growth, sporulation and conidial germination of the ATCC 74040 commercial strain of Beauveria bassiana. Mycelial growth was evaluated on potato dextrose agar plates with 100%, 75%, 50%, 25%, 12.5%, 6.25% and 0% of the recommended application rates of each fungicide at 15 and 25°C. Sporulation and conidial germination were determined at the recommended field doses of each fungicide at 25°C. All copper fungicides had fungistatic or fungicidal effects on B. bassiana that varied according to the dose. Only in two cases, copper oxide at 15°C and copper hydroxide at 25°C, at the lowest concentration of 6.5%, was mycelial growth not statistically significantly inhibited. Inhibition of mycelial growth depended both on the fungicide and its concentration, and partly on temperature. Both sporulation and conidial germination of B. bassiana were significantly inhibited by all fungicides. All fungicides inhibited the sporulation in a similar way (99.8%–100%). With the exception of copper oxychloride (inhibition, 13.6%), the other fungicides showed high detrimental effects on conidial germination (inhibition, 91.7%–100%). The fungus was strongly affected by some fungicides even at the lowest doses. The biological index used for the B. bassiana with copper fungicides ranged from 0.6 (copper sulphate) to 18.1 (copper oxychloride). Therefore, the tested fungicides were classified into the upper half of the highly toxic (T) category and are considered incompatible with the entomopathogenic fungus B. bassiana strain ATCC 74040 under in vivo experimental conditions. These results need to be further verified in vitro under both greenhouse and open-field conditions.     

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.