Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin.

The operon containing the genes encoding the subunits of the binary crystal toxin of Bacillus sphaericus strain LP1-G, BinA and BinB (41.9 kDa and 51.4 kDa, respectively), was cloned and sequenced. Purified crystals were not toxic to Culex pipiens larvae. Comparison of the amino-acid sequences of this strain (Bin4) with those of the three other known toxin types (Bin1, Bin2 and Bin3) revealed mutations at six positions, including a serine at position 93 of BinA4, whereas all other types of BinA toxin from B. sphaericus had a leucine at this position. Reciprocal site-directed mutagenesis was performed to replace this serine in BinA4 from LP1-G with a leucine and the leucine in the BinA2 protein from strain 1593 with a serine. Native and mutated genes were cloned and overexpressed. Inclusion bodies were tested on C. pipiens larvae. Unlike the native Bin4 toxin, the mutated protein was toxic, and the reciprocal mutation in Bin2 led to a significant loss of toxicity. In vitro receptor-binding studies showed similar binding behaviour for native and mutated toxins. In the absence of any experimental data on the 3D structure of these proteins, sequence analysis and secondary-structure predictions were performed. Amino acid 93 of the BinA polypeptide probably belongs to an alpha helix that is sensitive to amino-acid modifications. Position 93 may be a key element in the formation of the BinA-BinB complex responsible for the toxicity and stability of B. sphaericus Bin toxins.     

Mitochondrial uncoupling proteins and phylogenesis--UCP4 as the ancestral uncoupling protein

We searched for the previously defined uncoupling protein (UCP) signatures [Jezek, P. and Urbánková, E. (2000) IUBMB Life 49, 63-70] in genomes of Drosophila melanogaster, Caenorhabditis elegans, Dictyostelium discoideum, and Arabidopsis thaliana. We identified four UCPs in Drosophila and one in Caenorhabditis or Dictyostelium as close relatives of human UCP4 (BMCP), but distant from UCP1, UCP2, UCP3, and two plant UCPs of Arabidopsis. But the third Arabidopsis UCP is the closest UCP4 relative. This suggests that UCP4 represents the ancestral UCP from which other mammalian and plant UCPs diverged. Speculations on UCP4 participation in apoptosis are thus supported by its early phylogenetic occurrence.     

Hybrid peptide-polyketide natural products: biosynthesis and prospects toward engineering novel molecules

The structural and catalytic similarities between modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) inspired us to search for hybrid NRPS-PKS systems. By examining the biochemical and genetic data known to date for the biosynthesis of hybrid peptide-polyketide natural products, we show (1) that the same catalytic sites are conserved between the hybrid NRPS-PKS and normal NRPS or PKS systems, although the ketoacyl synthase domain in NRPS/PKS hybrids is unique, and (2) that specific interpolypeptide linkers exist at both the C- and N-termini of the NRPS and PKS proteins, which presumably play a critical role in facilitating the transfer of the growing peptide or polyketide intermediate between NRPS and PKS modules in hybrid NRPS-PKS systems. These findings provide new insights for intermodular communications in hybrid NRPS-PKS systems and should now be taken into consideration in engineering hybrid peptide-polyketide biosynthetic pathways for making novel "unnatural" natural products.     

Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals

The expression of metal-binding proteins or peptides in microorganisms and plants in order to enhance heavy metal accumulation and/or tolerance has great potential. Several different peptides and proteins have been explored. This review focuses on cadmium (Cd) because of the significant importance of this metal and because of its global presence in many food materials.     

African swine fever virus IAP homologue inhibits caspase activation and promotes cell survival in mammalian cells

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.     

Preparation of asymmetric,cerebroside sulfate-containing phospholipid vesicles

A procedure is described which inserts asymmetrically cerebroside sulfate (‘sulfatide’) into the outer leaflet of bilayered phospholipid vesicles. Cerebroside sulfate is adsorbed onto a cellulose, filter-paper support and, when incubated with phosphatidylcholine vesicles is transferred to and inserted into the outer leaflet of these vesicles. This transfer occurs at, or above the transition temperature of the phospholipid and follows a similar pattern with small or larger (‘fused’) unilamellar vesicles. The transfer is linear with time for 1–2 h and is maximal after about 6 h, when the sulfatide content reaches about 6 mol% of the total quantity of phospholipid, corresponding to about 10 mol% of the phospholipids present in the outer layer. Initial rates of sulfatide transfer were somewhat increased when the vesicles contained a positively charged lipid (e.g. stearylamine) and decreased when this lipid was negatively charged (e.g. dicetyl phosphate) or hydrophobic (e.g. cholesterol). Divalent ions markedly inhibited sulfatide transfer and monovalent ions did so to a lesser degree. Once incorporated into the outer leaflet of the vesicle, the sulfatide could not be removed by washing with buffer, 1 M NaCl or 1 M urea.     

Induction of mutations inSerratia marcescens by a proteosynthesis block

The formation of colour mutations ofSerratia marcescens by the action of chloramphenicol was studied. Pink variants were the commonest; the proportion of white variants was much smaller. Almost 100% mutations were formed in a two-day culture containing 100 μg. chloramphenicol/ml. Comparative experiments showed that the change in pigment formation was hereditary, i.e. that actual mutation, and not selection of chloramphenicol-resistant mutants, occurred. Mutation occurred both in strain 151 and strain HY. The resultant mutations remained constant throughout ten passages on normal nutrient medium. The minimum chloramphenicol concentration which produced an increase in the mutation frequency was 5 μg./ml. The combined effect of X-ray irradiation and chloramphenicol treatment somewhat stimulated the increase in the frequency of mutation as compared with cells which were only irradiated. The increase in the frequency of mutation was much slower on solid medium containing chloramphenicol.     

The genus Candida Berkhout

The properties of 53 fermentation type II strains of the genusCandida Berkhout were studied. The strains in question were originally identified asCandida tropicalis (Castellani) Berkhout,Candida pelliculosa Redaelli,Candida robusta Diddens et Lodder,Candida intermedia (Cif. et Ashf.) Langeron et Guerra,Candida langeroni Dietrichson,Candida obtusa (Dietrichson) v. Uden et Carmo Sousa and as various intermediate forms between these and other similar species. The classification criteria were extended by a number of very important characteristics, such as the degree of utilization of raffinose, the assimilation of lysine, xylose, cellobiose, maltotriose, maltotetraose and arabinose, virulence for mice, nutrient requirements, serological properties, etc. Actual classification was based on the numerical method of a similarity count. On the basis of this extension of the classification criteria, the characteristics of the speciesCandida tropicalis (Castellani) Berkhout andCandida pelliculosa Redaelli were defined in greater detail.Candida intermedia, evaluated on the basis of previously employed characteristics (lactose utilization, non-assimilation of KNO₃) does not appear to be a separate species, but a collection of different border-line forms of other species of this group.Candida robusta Diddens et Lodder is regarded as a member of the genusSaccharomyces, notCandida. The varietiesCandida tropicalis var.lambica andCandida pelliculosa var.cylindrica likewise do not seem to belong to the species concerned and will have to be studied in greater detail from the genetic aspect, in relation to other membrane-forming types ofCandida. The authors' extension of the classification criteria considerably reduced intraspecific variability, particularly in the speciesCandida tropicalis (Castellani) Berkhout, and led to greater accuracy in the practical diagnosis of this species, which is frequent in clinical material.