To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of OK-432 (picibanil) on the estrogen receptors of MCF-7 cells and potentiation of antiproliferative effects of tamoxifen in combination with OK-432.

OK-432 (picibanil), a streptococcal preparation, has a strong biological response modifier (BRM) function and is expected to produce clinical improvement and prolongation of survival in treated cancer patients in Japan. We were interested in whether OK-432 augments estrogen receptor (ER) levels in breast cancer. To investigate the effect of the BRMs on cellular growth and the characteristics of ER and progesterone receptors (PgR) in the human breast cancer cell line MCF-7, we used OK-432, Krestin (PSK), a protein-bound polysaccharide extracted from Coriolus versicolor, and lentinan, a fungal branched (1...3)-beta-D-glycan. OK432 and PSK dose dependently inhibited DNA synthesis of MCF-7 cells, and the 50% inhibitory concentrations of OK-432 and PSK were 1.2 KE (klinische Einheit, clinical unit)/ml and 200 micrograms/ml, respectively. Lentinan showed no direct anticancer effect in vitro. We found that OK-432 induced a 2-fold increase in ER levels in MCF-7 cells at 0.005 KE/ml, but not in PgR. Lentinan and low-dose PSK did not change ER or PgR levels, but high-dose PSK decreased ER and PgR. We also studied the combined effect of OK-432 and antiestrogens, tamoxifen (TAM) and DP-TAT-59. The combined treatment with OK-432 and TAM showed an additive inhibitory effect on MCF-7 cells. These results suggest that OK-432 may augment the therapeutic effect of TAM in breast cancer.

Antineoplastic Agents↗

PubMedDate not supplied

Interaction of amino acid-substituted partial extension peptides of adrenodoxin precursor with artificial membranes and isolated mitochondria.

The N-terminal fragment Met-Ala-Ala-Arg-Leu-Leu-Arg-Val-Ala-Ser-Ala-Ala-Leu-Gly (PA1-14) of the adrenodoxin precursor was previously found to inhibit the import of the precursor into mitochondria. In order to obtain further information on the structure-function relationship, five analogs of PA1-14 ([Leu1]PA1-14 (1), [Leu1,Ala10]PA1-14 (2), [Leu1,Ser4]PA1-14 (3), [Leu1,Arg11]PA1-14 (4) and [Leu1,Ser7,Arg10]PA1-14 (5) were synthesized. The CD study showed that PA1-14 and all analogs were random in an aqueous solution and formed an alpha-helical structure in the solution containing acidic liposomes. Peptides 1 and 2 were found to cause dye leakage from lipid vesicles more strongly than other analogs and PA1-14. All analogs completely inhibited the import of the precursor into mitochondria at a concentration of 30 microM. However, 1 and 2 destroyed the mitochondrial membrane potential. These results indicate that an increase in hydrophobicity by replacement of the Met and Ser residues by Leu and Ala, respectively, participates in the perturbation of the membranes. Furthermore, the requirement for the number and position of the Arg residue was found to be not very strict, although its presence in the extension peptide is essential for the precursor to import into mitochondria.

1,2-Dipalmitoylphosphatidylcholine↗

PubMedDate not supplied

Definitive proton beam radiation therapy for inoperable gastric cancer: a report of two cases.

Proton beam radiation therapy using 250 MeV protons was carried out on two patients with early gastric cancer (T1, N0, M0). One patient was an 85-year-old man with early gastric cancer of type IIa + IIc. The other one was a 70 year old man with early gastric cancer of type IIc. In both cases histological examination of biopsy specimens showed differentiated adenocarcinoma; distant metastasis was not found by other examinations. Both patients were considered inoperable due to their poor cardiac and/or respiratory functions. Therefore, it was decided to treat them by definitive proton irradiation, delivering total doses of 86 Gy and 83 Gy, respectively. In both patients, skin erythema that did not require any special treatment was found in the irradiation field. Hematobiological examinations did not show any abnormality. Although endoscopic examination at two years after irradiation in the former case and at seven months in the latter case showed persistent gastric ulcer at the site of the cancerous lesions, cancer cells were not found histologically. Therefore, we concluded that proton irradiation therapy was useful for inoperable early gastric cancers.

Adenocarcinoma↗

PubMedDate not supplied

Effects of model extension peptides containing glycine, proline and serine residues on the import of mitochondrial enzyme precursors into mitochondria.

We found previously that Ac-(Leu-Leu-Ala-Arg-Leu)3-NHCH3 (5(3)), a model of the extension peptide of cytochrome P-450(SCC) precursor, strongly inhibited the import of the precursor into mitochondria. Unexpectedly, however, 5(3) showed the break of respiratory control of mitochondria. In the present study, desAc- [Pro7, Ser10] 5(3) (1), [Pro7, Ser10] 5(3) (2), desAc- [Gly5, Pro7, Ser10] 5(3) (3) and [Gly5, Pro7, Ser10] 5(3) (4) were synthesized in order to investigate the influence of the Gly, Pro and Ser residues, which are present in the extension peptide, on the import and respiration. CD measurement indicated that all the peptides had an alpha-helical conformation in the presence of DPPC-DPPG (3:1) liposomes. The order of the content of alpha-helical conformation was 5(3) greater than 1, 2 greater than 3, 4, while SEP1-15 (N-terminal 1-15 fragment of the extension peptide of cytochrome P-450(SCC) precursor) showed no alpha-helical structure. The measurements of dye-leakage from liposomes, import-inhibition of P-450(SCC) and adrenodoxin precursors and respiratory inhibition of mitochondria indicated that 3 and 4, rather than 1 and 2, are similar to SEP1-15, and that the glycine residue in the extension peptide is of considerable importance for the import of the precursors.

Amino Acid Sequence↗