Safety and antitumor activity of recombinant soluble Apo2 ligand.

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

Expression of heme oxygenase-1 in atherosclerotic lesions.

Heme oxygenase-1 (HO-1) is a heme-degradation enzyme induced under various oxidative stress conditions. To elucidate the potential involvement of HO-1 in atherogenesis, the expression of this enzyme in atherosclerotic lesions of apolipoprotein E-deficient mice and humans were examined. Both immunostaining and in situ hybridization clearly demonstrated that the expression of HO-1 was prominent in endothelium and foam cells/macrophages of thickened intima in lesions from both humans and experimental animals. The expression of this enzyme was also detected in medial smooth muscle cells of advanced lesions. The induction of HO-1 mRNA was observed in murine peritoneal macrophages after treatment with oxidized low density lipoprotein (LDL) but not with native LDL in a dose-dependent manner. Time course study demonstrated that the induction was prominent at 3 hours, reached a maximal induction at 6 hours, and remained evident up to 24 hours after oxidized LDL treatment. The degree of induction was in concordant with the extent of oxidation in the LDL preparation. Lysophosphatidylcholine, one of the major components present in oxidized LDL, was ineffective to induce the gene expression, suggesting that other lipophilic substances derived from LDL oxidation are responsible for the induction of HO-1. These results clearly demonstrate that HO-1 is one of the stress proteins expressed in atherosclerotic lesions.

Molecular evidence for the hybrid origin of Paulownia Taiwaniana based on RAPD markers and RFLP of chloroplast DNA.

Genomic DNA of Paulownia fortunei, P. kawakamii and P. taiwaniana were amplified with 10-base primers of arbitrary sequences using the polymerase chain reaction (PCR). A total of 351 DNA fragments were amplified from 23 primers and of these 265 fragments (75.5%) were polymorphic. Almost all of the PCR-amplified products of P. taiwaniana were shared by either P. fortunei or P. kawakamii, or both, and the number of polymorphic fragments shared by P. taiwaniana and P. fortunei was about equivalent to those shared by P. taiwaniana and P. kawakamii. Restriction fragments of chloroplast DNA (cpDNA) purified from Paulownia species and from reciprocal crosses between P. fortunei and P. kawakamii were analyzed. Restriction enzyme SalI-digested cpDNA showed an identical pattern in both P. kawakamii and P. taiwaniana. These results further support the hypothesis that P. taiwaniana is the natural hybrid between P. fortunei and P. kawakamii and that the maternal parent of P. taiwaniana is P. kawakamii.

Periplasmic secretion of human growth hormone by Escherichia coli.

The gene coding for human growth hormone (hGH) was fused to the coding sequence for the signal peptide of a secreted Escherichia coli protein. STII heat-stable enterotoxin. This hybrid gene was expressed in E. coli. The signal peptide is properly processed and hGH is secreted in to the periplasmic space. In E. coli, some of the material made is proteolytically clipped or deamidated. The effect of culture conditions on the expression and secretion of hGH was studied and several important parameters were identified, including culture temperature and duration, cultivation pH, K+ levels, plasmid structure, and nutrient supplements. Alteration of culture conditions significantly improves the recovery yield and product quality of human growth hormone.

Biosynthetic 20-kilodalton methionyl-human growth hormone has diabetogenic and insulin-like activities.

The anterior pituitary gland produces a 20-kilodalton (kDa) variant of human growth hormone (hGH) that differs from the predominant 22-kDa form of hGH in that amino acid residues 32-46 are deleted. Previous work has suggested that the 20-kDa variant possesses the full growth-promoting and lactogenic activities of 22-kDa hGH but lacks its intrinsic diabetogenic and insulin-like activities. In the present study, recombinant DNA techniques were used to prepare biosynthetic 20-kDa hGH, and some of the biological properties of the purified hGH variant were examined. The biosynthetic 20-kDa hGH variant was found to share the propensity for aggregation exhibited by its native counterpart. Moreover, like the native variant, biosynthetic 20-kDa hGH possessed full growth-promoting activity in the weight gain test in hypophysectomized rats. However, contrary to previous work suggesting that native 20-kDa hGH lacks diabetogenic and insulin-like activities, biosynthetic 20-kDa hGH was found to have substantial diabetogenic activity when administered chronically to ob/ob mice and to possess approximately 20% the in vitro insulin-like activity of biosynthetic 22-kDa hGH on isolated epididymal adipose tissue of hypophysectomized rats. The diabetogenic and insulin-like activities of biosynthetic 20-kDa hGH cannot be ascribed to contamination of the hormone preparation with the 22-kDa form of hGH or with other diabetogenic or insulin-like pituitary peptides. Therefore, the results strongly suggest that diabetogenic and insulin-like activities are also intrinsic properties of the 20-kDa variant of hGH.

An improved method for the preparation of intermediate-purity antihemophilic factor concentrate for therapeutic usage.

An improved method involving pH changes in the process for the preparation of intermediate-purity antihemophilic concentrate offers several advantages over the standard method. Antihemophilic concentrates thus prepared are more soluble, more purified and more stable than the standard antihemophilic concentrates. High-potency antihemophilic concentrates with good solubility may be obtained by concentrating the [in-process] solution before lyophilization.

Removal of anticomplementary activity from human immune serum globulins by treatment with purified human plasmin.

Human immune serum globulin preparations were treated with highly purified human plasmin to remove their anticomplementary activity. The extent of anticomplementary activity removed and the amount of fragmentation obtained from immune serum globulin preparation depended upon the amount of plasmin added and the length of incubation time. The enzymatic reaction was stopped by removing plasmin with bentonite adsorption. Two types of plasmin-treated immune serum globulin that may be used for intravenous injection were obtained. One was a more fragmented product (35 to 45%) with lower anticomplementary activity, the other was a less degraded product (15 to 20%) with slightly higher anticomplementary activity. Antibody content of the former preparation was somewhat reduced while that of the latter preparation was virtually unchanged from the untreated immune serum globulin.

Enzymic and immunochemical properties of lysozyme. XVI. A novel synthetic approach to an antigenic reactive site by direct linkage of the relevant conformationally adjacent residues constituting the site.

Previous studies from this laboratory on the immunochemistry of specific chemical derivatives of native lysozyme and of the two disulfide peptide 62-68 (Cys 64-Cys 80) 74-97 (Cys 76-Cys 94) (i.e. (SS)2-peptide), have established an antigenic reactive site to comprise the spatially contiguous surface residues: Trp 72, Lys 97, Lys 96, Asn 93, Thr 89 and Asp 87. In the present work, the identity of the site was verified by an entirely different and novel approach. The aforementioned amino acids were linked directly into a single linear peptide with an intervening spacer where appropriate and substituting phenylalanine for tryptophan (i.e. Phe-Gly-Lys-Asn-Thr-Asp). This peptide (which does not exist in native lysozyme but simulates a surface region of the protein) possessed a remarkable inhibitory activity towards the reaction of lysozyme with its antisera. The immunochemical reactivity of the peptide was equal to the maximum expected reactivity of the site (i.e. a third of the total antigenic reactivity of lysozyme). These findings define quite conclusively and accurately the reactive site which is clearly composed of spatially adjacent residues that are distant in sequence reacting as if in direct linear linkage. The unequivocal establishment of this concept indicates that antigenic sites need not always be composed of residues in direct peptide linkage in the sequence. The nature of the site may depend on the protein. This unorthodox attack at the problem provides a novel and powerful approach for final delineation of the antigenic reactive sites (and perhaps other types of binding sites) in native proteins, following the completion of accurate narrowing down by chemical methods.