Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis.

The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the VH domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire VH-coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.     

Structural studies of a human gamma 3 myeloma protein (Jir) bearing the allotypic marker Gm(st)

The authors established the amino acid substitutions determining G3m(s) and G3m(t) specificities, which characterize Mongoloid populations, by sequence analysis of the Fc region of a myeloma protein (Jir). By comparing the amino acid sequences of the IgG3 (Jir) and the other IgG subclasses analyzed to date, it was found that G3m(s) was an isoallotype specified by an amino acid substitution at position 435; i.e., whereas the subclasses IgG1, IgG2, and IgG4 had histidine in common, G3m(s-) had arginine in this position. This was also confirmed by the observation that the Fc fragment in question bound to protein A. It was also established that the amino acid at position 379 of G3m(t-) IgG3 and the other subclasses was valine, whereas methionine in this position was specific for G3m(t+). In addition, the amino acids at position 339 of G3m(u-) IgG3 Jir was threonine, and at position 296 of G3m(g-) IgG3 Jir was tyrosine. These findings are not in accord with the hitherto postulated relations of alanine and phenylalanine to G3m(u-) and G3m(g-), respectively. Finally, this study showed that a large number of substitutions occurred at positions 384 through 389, which suggests that many specificities of the G3m(b) group occur on IgG3 proteins.     

Activation of TIMP-2/progelatinase A complex by stromelysin.

Progelatinase A was purified as a complex with TIMP-2 from the conditioned medium of a human glioblastoma cell line. The TIMP-2/progelatinase complex was resistant to the activation by p-aminophenylmercuric acetic acid (APMA), and showed less than 10% of the activity of the TIMP-2-free active enzyme. When the complex was incubated with stromelysin in the presence of APMA, the 64-kDa progelatinase was effectively converted to the 57-kDa mature enzyme, increasing its gelatinolytic activity about 8-fold. These results suggest that stromelysin is a natural activator of TIMP-2-bound progelatinase A.     

Novel Nano-Sized MR Contrast Agent Mediates Strong Tumor Contrast Enhancement in an Oncogene-Driven Breast Cancer Model

The current study was carried out to test the potential of a new nanomaterial (Spago Pix) as a macromolecular magnetic MR contrast agent for tumor detection and to verify the presence of nanomaterial in tumor tissue. Spago Pix, synthesized by Spago Nanomedical AB, is a nanomaterial with a globular shape, an average hydrodynamic diameter of 5 nm, and a relaxivity (r₁) of approximately 30 (mM Mn)⁻¹ s⁻¹ (60 MHz). The material consists of an organophosphosilane hydrogel with strongly chelated manganese (II) ions and a covalently attached PEG surface layer. In vivo MRI of the MMTV-PyMT breast cancer model was performed on a 3 T clinical scanner. Tissues were thereafter analyzed for manganese and silicon content using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The presence of nanomaterial in tumor and muscle tissue was assessed using an anti-PEG monoclonal antibody. MR imaging of tumor-bearing mice (n = 7) showed a contrast enhancement factor of 1.8 (tumor versus muscle) at 30 minutes post-administration. Contrast was retained and further increased 2–4 hours after administration. ICP-AES and immunohistochemistry confirmed selective accumulation of nanomaterial in tumor tissue. A blood pharmacokinetics analysis showed that the concentration of Spago Pix gradually decreased over the first hour, which was in good agreement with the time frame in which the accumulation in tumor occurred. In summary, we demonstrate that Spago Pix selectively enhances MR tumor contrast in a clinically relevant animal model. Based on the generally higher vascular leakiness in malignant compared to benign tissue lesions, Spago Pix has the potential to significantly improve cancer diagnosis and characterization by MRI.     

Physiological and Genomic Features of a Novel Sulfur-Oxidizing Gammaproteobacterium Belonging to a Previously Uncultivated Symbiotic Lineage Isolated from a Hydrothermal Vent

Strain Hiromi 1, a sulfur-oxidizing gammaproteobacterium was isolated from a hydrothermal vent chimney in the Okinawa Trough and represents a novel genus that may include a phylogenetic group found as endosymbionts of deep-sea gastropods. The SSU rRNA gene sequence similarity between strain Hiromi 1 and the gastropod endosymbionts was approximately 97%. The strain was shown to grow both chemolithoautotrophically and chemolithoheterotrophically with an energy metabolism of sulfur oxidation and O₂ or nitrate reduction. Under chemolithoheterotrophic growth conditions, the strain utilized organic acids and proteinaceous compounds as the carbon and/or nitrogen sources but not the energy source. Various sugars did not support growth as a sole carbon source. The observation of chemolithoheterotrophy in this strain is in line with metagenomic analyses of endosymbionts suggesting the occurrence of chemolithoheterotrophy in gammaproteobacterial symbionts. Chemolithoheterotrophy and the presence of homologous genes for virulence- and quorum sensing-related functions suggest that the sulfur-oxidizing chomolithotrophic microbes seek animal bodies and microbial biofilm formation to obtain supplemental organic carbons in hydrothermal ecosystems.     

Experimental Models for Prevention of Graft-versus-Host Reaction in Bone Marrow Transfusion

Induction and suppression of splenomegaly and cytotoxicity against C57BL/6 cells were studied in (AKR × C57BL/6) F1 hybrid adult mice after the transfer of AKR lymphoid and bone marrow cells. 1) Splenomegaly and cytotoxicity were dissociated in the developmental stages of the graft-versus-host reaction. When lymphoid and bone marrow cells of normal AKR mice were injected into F1 recipients, splenomegaly was prominent on days 5 and 7, but cytotoxicity of spleen cells was not detected. Splenomegaly became less prominent but the cytotoxicity became detectable on day 14 after the injection. 2) Cytotoxic activity of spleen cells of F1 recipients was suppressed by the treatment of AKR donors with C57BL/6 lymphoid cells in Freund's complete adjuvant. Splenomegaly, however, was substantially enhanced by such a treatment of the donors. On the other hand, induction of the cytotoxic activity was facilitated by the treatment of donors with C57BL/6 skin grafts. 3) F1 hybrid mice could be protected from the graft-versus-host reaction by the injection of AKR anti-C57BL/6 serum or pretreatment of AKR donors with sonicated cellular antigens of C57BL/6.