Monoclonal antibody A7 coupled to magnetic particles as a contrast enhancing agent for magnetic resonance imaging of human colorectal carcinoma

Background: Local recurrence, the most frequent pattern of recurrence of rectal carcinoma, is almost always fatal. The difficulty of diagnosing local recurrence contributes importantly to the poor prognosis. Methods: We coupled monoclonal antibody (Mab) A7, which reacts specifically with human colorectal carcinoma, to ferromagnetic lignosite (FML) particles to distinguish rectal carcinoma from other tissues by magnetic resonance (MR) imaging. We examined retention of immunoreactivity by the A7-FML complexes in vitro, and also their distribution in vivo according to radiolabeling and MR imaging when injected into nude mice bearing human colorectal carcinoma xenografts. Results: A7-FML retained binding activity nearly identical to that of Mab A7. Significantly more ¹²⁵I-labeled A7-FML accumulated in engrafted tumors than did ¹²⁵I-labeled normal mouse IgG-FML complexes (P<0.05). A7-FML disappeared rapidly from the blood. Normal tissues accumulated less ¹²⁵I-labeled A7-FML than tumors; this accumulation decreased linearly with time. In MR imaging, signal intensity was reduced in the tumor by the injection of A7-FML. Conclusions: A7-FML is potentially useful as a MR contrast enhancing agent for human colorectal carcinoma xenografts implanted subcutaneously.     

'Click chemistry' on polysaccharides: a convenient, general, and monitorable approach to develop (1-->3)-beta-D-glucans with various functional appendages

(1-->3)-beta-D-Glucans having various functional appendages (lactoside, ferrocene, pyrene, and porphyrin) can be prepared in an convenient, quantitative, and regioselective manner through regioselective bromination-azidation of curdlan to afford 6-azido-6-deoxycurdlan followed by chemoselective [3+2]-cycloadditions with various functional modules bearing a terminal alkyne group. The ability to monitor reaction conversions is an additional advantage of this synthetic approach over the conventional direct modifications on polysaccharides; the reaction can be readily monitored based on the intensity of azido peaks in the in situ attenuated total reflection infrared spectra.     

A novel combined thermometric and amperometric biosensor for lactose determination based on immobilised cellobiose dehydrogenase

A novel method for lactose determination in milk is proposed. It is based on oxidation of lactose by cellobiose dehydrogenase (CDH) from the basidiomycete Phanerochaete chrysosporium, immobilised in an enzyme reactor. The reactor was prepared by cross-linking CDH onto aminopropyl-silanised controlled pore glass (CPG) beads using glutaraldehyde. The combined biosensor worked in flow injection analysis (FIA) mode and was developed for simultaneous monitoring of the thermometric signal associated with the enzymatic oxidation of lactose using p-benzoquinone as electron acceptor and the electrochemically generated current associated with the oxidation of the hydroquinone formed. A highly reproducible linear response for lactose was obtained between 0.05 mM and 30 mM. For a set of more than 500 samples an R.S.D. of less than 10% was achieved. The assay time was ca. 2 min per sample. The sensor was applied for the determination of lactose in dairy milk samples (milk with a fat content of 1.5% or 3% and also "lactose free" milk). No sample preparation except dilution with buffer was needed. The proposed method is rapid, suitable for repeated use and allows the possibility to compare results from two different detection methods, thus providing a built-in quality assurance. Some differences in the response observed between the methods indicate that the dual approach can be useful in mechanistic studies of redox enzymes. In addition, a dual system opens up interesting possibilities for studies of enzyme properties and mechanisms.     

High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.     

Modulation of redox potential and alteration in reactivity via the peroxide shunt pathway by mutation of cytochrome P450 around the proximal heme ligand

In the thermophilic cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st), a phenylalanine residue at position 310 and an alanine residue at position 320 are located close to the heme thiolate ligand, Cys317. Single site-directed mutants F310A and A320Q and double mutant F310A/A320Q have been constructed. All mutant enzymes as well as wild-type (WT) P450st were expressed at high levels. The substitution of F310 with Ala and of A320 with Gln induced shifts in redox potential and blue shifts in Soret absorption of ferrous-CO forms, while spectral characterization showed that in the resting state, the mutants almost retained the structural integrity of the active site. The redox potential of the heme varied as follows: -481 mV (WT), -477 mV (A320Q), -453 mV (F310A), and -450 mV (F310A/A320Q). The trend in the Soret band of the ferrous-CO form was as follows: 450 nm (WT) < 449 nm (A320Q) < 446 nm (F310A) < 444 nm (F310A/A320Q). These results established that the reduction potential and electron density on the heme iron are modulated by the Phe310 and Ala320 residues in P450st. The electron density on the heme decreases in the following order: WT > A320Q > F310A > F310A/A320Q. The electron density on the heme iron infers an essential role in P450 activity. The decrease in electron density interferes with the formation of a high-valent oxo-ferryl species called Compound I. However, steady-state turnover rates of styrene epoxidation with H2O2 show the following trend: WT approximately equal to A320Q < F310A approximately equal to F310A/A320Q. The shunt pathway which can provide the two electrons and oxygen required for a P450 reaction instead of NAD(P)H and dioxygen can rule out the first and second heme reduction in the catalytic process. Because the electron density on the heme iron might be deeply involved in the k cat values in this system, the intermediate Compound 0 which is the precursor species of Compound I mainly appears to participate dominantly in epoxidation with H2O2.     

Oxidative stress is associated with aluminum toxicity recovery in apex of pea root

Aims

Although many studies on the mechanism of Al toxicity and tolerance have been conducted independently, events occurring during the recovery process from Al injury is limited. This study was to investigate Al toxicity recovery mechanism focusing in morphological and physiological aspect.

Methods

We investigated the mechanisms underlying Al toxicity recovery in terms of oxidative stress using the pea root apex as a model system.

Results

The accumulation of reactive oxygen species was remarkably high in the root under continued Al treatment but decreased in the recovering root. The superoxide anion exuded in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) showed a similar tendency with respect to the accumulation of reactive oxygen species. A similar pattern of lignin content and superoxide dismutase activity was observed among the treatments, while the increased peroxidation in the root under continued Al treatment did not decline with recovery treatment. A longitudinal section of the root under continued Al treatment showed the accumulation of superoxide anion, lignin and peroxide (H₂O₂) at the epidermal and outer cortex region where the Al induced injuries, including ruptures, are detected.

Conclusions

Oxidative stress is associated with the mechanism of Al toxicity recovery. The recovery process might include the elongation of the central cylinder as a consequence of the oxidative stress-induced formation of the zonal region (ZR). The results further suggest a plausible role for the ZR in the programmed cell death-like function involved in Al toxicity recovery.