Crystal structure of the antigen-binding fragment of apoptosis-inducing mouse anti-human Fas monoclonal antibody HFE7A.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A), which induces apoptosis, has been humanized based on a structure predicted by homology modeling. A version of humanized HFE7A is currently under development for the treatment of autoimmune diseases such as rheumatoid arthritis. For a deeper understanding of the protein engineering aspect of antibody humanization, for which information on the three-dimensional structure is essential, we determined the crystal structure of the m-HFE7A antigen-binding fragment (Fab) by X-ray crystallography at 2.5 A resolution. The main-chain conformation of the five loops in the six complementarity-determining regions (CDRs) was correctly predicted with root-mean-square deviations of 0.30-1.04 A based on a comparison of the crystal structure with the predicted structure. The CDR-H3 conformation of the crystal structure, which was not classified as one of the canonical structures, was completely different from that of the predicted structure but adopted the conformation which followed the "H3-rules." The results of charge distribution analysis of the antigen-binding site suggest that electrostatic interactions may be important for its binding to Fas.     

Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of 20(R),22(R)-dihydroxycholesterol and reduced adrenodoxin

Electron paramagnetic resonance (EPR) spectra of ferrous-nitric oxide (14NO and 15NO) cytochrome P-450scc complexed with 20(R),22(R)-dihydroxycholesterol were measured at 77 K with X-band (9.35 GHz) microwave frequency. The EPR spectra clearly showed the spin system to have rhombic symmetry (gx = 2.068, gz = 2.001, gy = 1.961, and Az = 1.89 mT for 14NO) and were distinct from those of 20(S)-hydroxycholesterol complexes. The unique nature of the 20(S)-hydroxycholesterol complexes indicates that 20(S)-hydroxycholesterol is not a proper intermediate in the cholesterol side-chain cleavage reaction. In addition, among various steroid complexes of ferrous-NO species having rhombic symmetry, the EPR spectra of 20(R),22(R)-dihydroxycholesterol complexes were significantly different from those of 22(R)-hydroxycholesterol complexes, suggesting that upon 20S-hydroxylation of 22(R)-hydroxycholesterol the conformation of the active site changes so as to facilitate subsequent cleavage of the C20-C22 bond of the cholesterol side chain. Addition of reduced adrenodoxin to the ferrous-NO cytochrome P-450scc complex in the presence of cholesterol caused a complete shift of the gx = 2.070 signal to gx = 2.075, indicating a reorientation of cholesterol in the substrate-binding site of the enzyme upon adrenodoxin binding. Without reduced adrenodoxin, the process of reorientation of cholesterol in the substrate-binding site was very slow, requiring more than 50 h of incubation at 0 degrees C. The present observations suggest that adrenodoxin may have another positive role in the cholesterol side-chain cleavage reaction, in addition to transferring an electron to the heme of cytochrome P-450scc.     

Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues.

P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.     

Changes in K+,Na+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

A homodimer protein consisting of two 38,000 dalton peptides was isolated from a murine leukemia cell line (M1). The binding molar ratio of the 38K-dimer protein to purified skeletal muscle actin was saturated at 1:3, and when the 38K-dimer/actin ratio exceeded 1:12, gelation occurred. This gelation was completely inhibited by the presence of either 10 mM KCl or 20 mM NaCl. The protein induced actin filament bundling, which required a higher 38K-dimer/actin ratio and was not affected by the presence of monovalent cations. During the differentiation of Ml cells, the sensitivity of the 38K protein to monovalent cations was decreased; that is 20 mM KCl or 50 mM NaCl was required to inhibit the gelation by the 38K protein isolated from differentiated cells. On the other hand, the intracellular K+ content of Ml cells decreased from 70 +/- 5 mM to 18 +/- 3 mM, and Na+ increased from 10 +/- 5 mM to 40 +/- 10 mM during the differentiation. These findings suggest that the differentiation brought about conditions favourable for the 38K protein to induce actin gelation, and in turn, the locomotive and phagocytic activities which were induced only after differentiation in this cell line.     

Transgene expression in mammary glands of newborn rats

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals. © 1996 Wiley-Liss, Inc.     

Macrophage-Specific Overexpression of Human Matrix Metalloproteinase-12 in Transgenic Rabbits

Increased matrix metalloproteinase-12 (MMP-12) has been implicated in atherosclerosis and many other inflammatory processes. To define MMP-12 functions in vivo, we generated transgenic rabbits that expressed human (h) MMP-12 gene under the control of a macrophage-specific promoter, the human scavenger receptor promoter. Two transgenic founder rabbits were found to have hMMP-12 transgene integration by Southern blot analysis. hMMP-12 mRNA was expressed in peritoneal and alveolar macrophages, and in tissues enriched in macrophages in transgenic rabbits. High levels of hMMP-12 protein were detected in the conditioned media of cultured peritoneal and alveolar macrophages from transgenic rabbits. Zymography showed that hMMP-12 secreted from macrophages possessed enzymatic activity toward β-casein. To evaluate the expression of hMMP-12 in inflammatory sites, we used carrageenan-induced granulomas as an in vivo model for tissue macrophages and foam cells. Granuloma size in transgenic rabbits was significantly increased compared to that in control rabbits, and histological examination revealed that granulomas of transgenic rabbits were enriched in macrophages associated with increased hMMP-12 expression. We believe that this transgenic rabbit model with increased expression of hMMP-12 may become a useful model for further mechanistic studies of MMP-12 in inflammatory diseases and cancer invasion; it is also an ideal model for testing the in vivo action of MMP-12 inhibitors.     

Overexpression of a rice gene encoding a small C2 domain protein OsSMCP1 increases tolerance to abiotic and biotic stresses in transgenic Arabidopsis

Plant growth and crop production are limited by environmental stress. We used a large population of transgenic Arabidopsis expressing rice full-length cDNAs to isolate the rice genes that improve the tolerance of plants to environmental stress. By sowing T2 seeds of the transgenic lines under conditions of salinity stress, the salt-tolerant line R07047 was isolated. It expressed a rice gene, OsSMCP1, which encodes a small protein with a single C2 domain, a Ca²⁺-dependent membrane-targeting domain. Retransformation of wild-type Arabidopsis revealed that OsSMCP1 is responsible for conferring the salt tolerance. It is particularly interesting that R07047 and newly constructed OsSMCP1-overexpressing Arabidopsis showed enhanced tolerance not only to high salinity but also to osmotic, dehydrative, and oxidative stresses. Furthermore, R07047 showed improved resistance to Pseudomonas syringae. The OsSMCP1 expression in rice is constitutive. Particle-bombardment-mediated transient expression analysis revealed that OsSMCP1 is targeted to plastids in rice epidermal cells. It induced overexpression of several nuclear encoded genes, including the stress-associated genes, in transgenic Arabidopsis. No marked morphological change or growth retardation was observed in R07047 or retransformants. For molecular breeding to improve the tolerance of crops against environmental stress, OsSMCP1 is a promising candidate.     

Thirteen novel deoxynivalenol-degrading bacteria are classified within two genera with distinct degradation mechanisms

The mycotoxin deoxynivalenol (DON), a secondary metabolite produced by species of the plant pathogen Fusarium, causes serious problems in cereal crop production because of its toxicity towards humans and livestock. A biological approach for the degradation of DON using a DON-degrading bacterium (DDB) appears to be promising, although information about DDBs is limited. We isolated 13 aerobic DDBs from a variety of environmental samples, including field soils and wheat leaves. Of these 13 strains, nine belonged to the Gram-positive genus Nocardioides and other four to the Gram-negative genus Devosia. The degradation phenotypes of the two Gram types were clearly different; all washed cells of the 13 strains degraded 100 μg mL(-1) DON to below the detection limit (0.5 μg mL(-1)), but the conditions inducing the DON-degrading activities differed between the two Gram types. The HPLC profiles of the DON metabolites were also distinct between the two genera, although all strains produced 3-epi-deoxynivalenol. The Gram-positive strains showed DON assimilation in media containing DON as a carbon source, whereas the Gram-negatives did not. Our results suggest that aerobic DDBs are distributed within at least two phylogenetically restricted genera, suggesting independent evolution of the DON-degradation mechanisms.     

Characterization of a Novel Plasmid,pMAH135, from Mycobacterium avium Subsp. hominissuis

Mycobacterium avium complex (MAC) causes mainly two types of disease. The first is disseminated disease in immunocompromised hosts, such as individuals infected by human immunodeficiency virus (HIV). The second is pulmonary disease in individuals without systemic immunosuppression, and the incidence of this type is increasing worldwide. M. avium subsp. hominissuis, a component of MAC, causes infection in pigs as well as in humans. Many aspects of the different modes of M. avium infection and its host specificity remain unclear. Here, we report the characteristics and complete sequence of a novel plasmid, designated pMAH135, derived from M. avium strain TH135 in an HIV-negative patient with pulmonary MAC disease. The pMAH135 plasmid consists of 194,711 nucleotides with an average G + C content of 66.5% and encodes 164 coding sequences (CDSs). This plasmid was unique in terms of its homology to other mycobacterial plasmids. Interestingly, it contains CDSs with sequence homology to mycobactin biosynthesis proteins and type VII secretion system-related proteins, which are involved in the pathogenicity of mycobacteria. It also contains putative conserved domains of the multidrug efflux transporter. Screening of isolates from humans and pigs for genes located on pMAH135 revealed that the detection rate of these genes was higher in clinical isolates from pulmonary MAC disease patients than in those from HIV-positive patients, whereas the genes were almost entirely absent in isolates from pigs. Moreover, variable number tandem repeats typing analysis showed that isolates carrying pMAH135 genes are grouped in a specific cluster. Collectively, the pMAH135 plasmid contains genes associated with M. avium’s pathogenicity and resistance to antimicrobial agents. The results of this study suggest that pMAH135 influence not only the pathological manifestations of MAC disease, but also the host specificity of MAC infection.