Quantitative patterns of the thermal inactivation of microorganisms as a basis for obtaining high-quality vaccinal preparations

The model of the thermal inactivation of microorganisms is presented. This model permits the explanation of all existing kinetic dependencies relating to the death of microorganisms, the restoration of sublethal damages, the influence of the medium and the temperature of incubation on the process of the production and control of vaccinal preparations.     

Involvement of Azopirillum brasilense plasmid DNA in the productio of indole acetic acid

Indole acetic acid (IAA) production in Azospirillum brasilense strain Sp245 is controlled by a 85 MDa plasmid naturally present in this bacterium. In the presence of L-tryptophan, anthranilic acid production and almost no IAA production occurs in a derivative strain harbouring a Tn5-Mob insertion in the 85 MDa plasmid. Agrobacterium tumefaciens strain GM19023, upon transfer of Tn5-Mob labelled 85 MDa plasmid of A. brasilense Sp245, gains the ability to produce anthranilic acid.     

Controlling the near‐infrared transparency of costal cartilage by impregnation with clearing agents and magnetite nanoparticles

Penetration depth of near‐infrared laser radiation to costal cartilage is controlled by the tissue absorption and scattering, and it is the critical parameter to provide the relaxation of mechanical stress throughout the whole thickness of cartilage implant. To enhance the penetration for the laser radiation on 1.56 μm, the optical clearing solutions of glycerol and fructose of various concentrations are tested. The effective and reversible tissue clearance was achieved. However, the increasing absorption of radiation should be concerned: 5°C‐8°C increase of tissue temperature was detected. Laser parameters used for stress relaxation in cartilage should be optimized when applying optical clearing agents. To concentrate the absorption in the superficial tissue layers, magnetite nanoparticle (NP) dispersions with the mean size 95 ± 5 nm and concentration 3.9 ± 1.1 × 10¹¹ particles/mL are applied. The significant increase in the tissue heating rate was observed along with the decrease in its transparency. Using NPs the respective laser power can be decreased, allowing us to obtain the working temperature locally with reduced thermal effect on the surrounding tissue.      

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine – a transition state analog of non-specific substrate

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1’-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1’-subsite.     

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr³⁺ ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr³⁺ ion binding mainly to N₇ (G) and to phosphate groups was demonstrated. ψ‐Type VCD spectra resembling electronic CD (ECD) spectra, which appear during ψ‐type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large‐scale helicity. Atomic force microscopy confirmed DNA condensation by Cr³⁺ ions and the formation of tight DNA particles responsible for the ψ‐type VCD spectra. Upon increasing the Cr³⁺ ion concentration the shape of the condensates changed from loose flower‐like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr³⁺ ions studied. The secondary structure of DNA remained in a B‐form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles. © 2002 Wiley Periodicals, Inc. Biopolymers 61: 243–260, 2002     

Immunogenicity of synthetic conjugates of Lewisy oligosaccharide with proteins in mice: towards the design of anticancer vaccines

 Many human carcinomas overexpress the Lewis^y (Le^y) blood-group epitope [Fucα1→2Galβ1→4 (Fucα1→3)GlcNAcβ1→3Gal-]. With a view to developing Le^y based vaccines we have examined the immunogenicity of Le^y-protein conjugates in mice. Le^y pentasaccharide was synthesized as its allyl glycoside and coupled to keyhole limpet hemocyanin (KLH) by reductive amination or by a novel method utilizing a maleido-derivitized alkyl carboxyhydrazide as a bridging group to 2-iminothiolane-derivitized KLH. Le^y oligosaccharide was also coupled to bovine serum albumin by reductive amination. Immunization of groups of mice with the three conjugates, together with the immunological adjuvant QS21, showed that Le^y oligosaccharide directly coupled to KLH was the most efficient conjugate for eliciting IgG and IgM antibody responses to naturally occurring forms of Le^y epitopes carried on mucins and glycolipids. These antibodies were also reactive with and cytotoxic to a human breast cancer cell line expressing Le^y (MCF-7). These experiments suggest that Le^y-KLH antigen and QS21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma patients. Received: 28 March 1997 / Accepted: 2 September 1997