Epitope Mapping of Human Alpha-Fetoprotein

The epitope structure of human alpha-fetoprotein (AFP) was studied using more than 50 monoclonal antibodies (MAB) to human AFP. These MAB obtained from various world laboratories of the TD-2 AFP Workshops of the International Society for Oncodevelopmental Biology and Medicine (ISOBM-1996-1998-2000) were analyzed by competitive immunoaffinity electrochromatography (IAE) on nitrocellulose membranes (NCM). Five types of interaction of the AFP–MAB complex with the MAB fixed on NCM were found: 1) complete neutralization; 2) partial neutralization; 3) unidirectional neutralization; 4) enhanced binding; 5) lack of interaction. By IAE, 51 MAB were found to recognize 23 different epitopes in the AFP molecule. Based on these findings, an epitope map of AFP was designed which consists of eight epitope clusters and eight individual epitopes. The epitope location is considered with respect to the conformational state of the AFP molecule. Possible causes of the five types of interaction found on neutralization are discussed.     

Structure of α-α-Hairpins with Short Connections in Globular Proteins

The analysis of conformations of more than 100 --hairpins with closely packed helical segments and connections up to four amino acid residues in length was carried out. Five types of the connections were revealed, and their and values on the Ramachandran map were found. Each type of --hairpins was shown to have a unique sequence pattern for hydrophobic and hydrophilic residues.     

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

A selective protonation strategy is described that uses [3-2H] 13C -ketoisovalerate to introduce (1H- methyl)-leucine and (1H- methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of 100 mg/L -ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] -ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.     

A low-temperature-responsive translation elongation factor 1α from barley (Hordeum vulgare L.)

A cDNA clone (pBLT63) encoding a protein synthesis elongation factor 1 (EF-1) was isolated from a low-temperature winter barley shoot meristem library by differential screening. The nucleotide sequence of the coding region of the low-temperature-induced barley gene shows very high homology with two EF-1 plant genes from tomato and Arabidopsis. The barley genome contains an EF-1 gene family situated on the short arm of chromosome 2 and the long arm of chromosome 5. The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Z23130.     

Evidence for a non-antioxidant,dose-dependent role of α -lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as -lipoic acid and -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with -lipoic acid and -tocopherol, alone or in combination, prior to incubation with H₂O₂ or staurosporine. -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H₂O₂ and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with -lipoic acid and/or -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with -lipoic acid and -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of -lipoic acid as an antioxidant.     

Effects of natural interferon α, natural tumor necrosis factor α and their combination on human mesothelioma xenografts in nude mice

Effects of human natural interferon (nIFN) alone, human natural tumor necrosis factor (nTNF) alone and their combination (OH-1) were tested on three human mesothelioma lines implanted in nude mice. Tumors were transplanted subcutaneously by trocar on treatment day –12. nIFN was given intraperitoneally (i.p.) at a dose of 2 × 10⁷ or 2 × 10⁸ IU kg^–1 day^–1, 5 days a week for 3 weeks. nTNF was given i.p. at a dose of 2 × 10⁷ or 2 × 10⁸ U kg^–1 day^–1 in the same schedule as that of nIFN. Tumor diameters were serially measured and tumor volumes were calculated. Antitumor effects were assessed by two methods: comparison of final tumor volumes in treated and control groups (T/C), and changes in median average total tumor volume. The treatment produced no clinically discernible toxicities. nIFN had strong inhibitory activity against all three human mesothelioma lines. nTNF alone had modest activity only at the high dose used. The combination of the two produced activity essentially similar to that produced by nIFN alone. High-dose nIFN may have a role as an active agent in the treatment of patients with mesothelioma.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Phase I study of TNFα AutoVaccIne in Patients with metastatic cancer

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNF in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNF proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNF antibodies. Secondary objectives were improvements in body weight and tumour response. Thirty-three patients were vaccinated with three doses (20, 100, or 400 g) of TNF vaccine at 2-weekly intervals adjuvanted with aluminium hydroxide. Anti-TNF antibody titres were measured by both a RIA, using soluble native TNF as the antigen, and by an ELISA using immobilized partly denatured TNF. Eleven patients (33%) had mild grade1/2 injection site reactions at the higher doses. In 10 of 20 patients, serum antibodies recognize denatured TNF in the ELISA, whereas, antibody titres against native TNF in the RIA were undetectable. This suggests that the production process had partly denatured the vaccine preventing the formation of cross-reacting antibodies to native TNF. In conclusion, TNF vaccine was able to elicit vaccine specific antibodies. However, since the antibodies were only able to cross-react with partly denatured TNF, evaluation of safety and tumour responses to the TNF vaccine was compromised.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Several Fmoc-,-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satisfactory.     

Relationship between soluble tumor necrosis factor (TNF) receptors and TNFα during immunotherapy with interleukin-2 and/or interferon α

Eleven metastatic cancer patients were studied during three different regimens of immunotherapy with interleukin-2 (IL-2) and/or interferon (IFN): group A received 4 days of IL-2 i.a. infusion (n=3), group B IFN s.c. during 5 days (n=4), followed on day 3 by 5 days of a continuous IL-2 i.v. infusion, and group C had 4 days of IL-2 i.v. infusion together with s.c. IFN on days 1 and 4 (n=4). Soluble tumor necrosis factor receptors (sTNFR) p55 and p75 and TNF concentrations in serum were analyzed before therapy and daily during 8 days of the first therapy cycle. sTNFR was measured by radioimmunoassay. sTNFR p55 increased in all patient groups from a baseline value of 5.2±0.9 ng/ml to a maximum of 13.6±1.2 ng/ml by days 3–4 (P=0.003). sTNFR p75 increased from 7.6±1.1 ng/ml to peak values of 30.1±2.6 ng/ml in groups A and B (P=0.02). In group C the sTNFR p75 response was weak (NS). In group B, the increase of both p55 and p75 occurred only after addition of IL-2 to IFN. TNF increased weakly during treatment with IFN alone (group B); it rose strongly during IL-2 and the combined treatment (groups A-C) from 8±2 pg/ml to 115±13 pg/ml (P=0.003). In group B, it reached the maximum 24 h after addition of IL-2 to IFN and decreased thereafter. there was a significant relationship between TNF and sTNFR p55 or sTNFR p75 in groups A and C, (P=0.001), but not in group B. Group C was also investigated during the third therapy cycle. The increase of sTNFR p75 was stronger (P=0.01) and that of TNF weaker than in the first cycle; the sTNFR p55 response was similar in both cycles. In conclusion sTNFR p55 and p75 are rapidly induced during IL-2 and IL-2+IFN treatment, the increase of sTNF receptors parallels or exceeds that of TNF and may influence the immunomodulatory effects of TNF during cytokine therapy.