Elevated serum α-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to α-fetoprotein

This study was carried out to clarify the reason for elevation of serum α-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.     

Disturbed Purkinje cell migration due to reduced expression of Reelin by X-irradiation in developing rat cerebellum.

The major histogenetic events of the rat cerebellum take place in the early postnatal days. During this period, precursors of microneurons, such as granule cells, form the external granular layer (EGL), extend over the surface of the primordial cerebellum, and actively proliferate. Postmitotic granule cells leave the EOL and migrate to the internal granular layer (IGL). On the other hand, guided by radial glial fibers, immature Purkinje cells migrate from the ventricular zone of the fourth ventricle and settle in the Purkinje cell plate with thickness of several cells. Various cell adhesion molecules are involved in the interaction between the migratory immature Purkinje cells and processes of the radial glia as the basis for contact guidance. The second process is the formation of immature Purkinje cells to the monolayer. This process takes place at the first week after birth of the rat and cell adhesion molecules such as neural cell adhesion molecule (NCAM), fibronectin, tenascin and Reelin are also suggested to play an important role for the cell patterning. When rat fetuses are exposed to X-radiation in the last gestation period, abnormal foliation of the cerebellum develops with ectopic Purkinje cells. The molecular mechanism that contributes to abnormal migration of Purkinje cells and foliar malformation induced by X-irradiation in the cerebellum are not yet clear. This study was undertaken to elucidate the mechanisms of ectopic Purkinje cell formation by examining the expression of cell adhesion molecules.     

Heme-binding properties of heme detoxification protein from Plasmodium falciparum

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.     

In vitro effects of bovine lactoferrin on autoaggregation ability and surface hydrophobicity of bifidobacteria

Changes in autoaggregation ability and surface hydrophobicity of bifidobacteria with addition of bovine lactoferrin in liquid media were investigated. Lactoferrin addition caused loss of autoaggregation ability, disappearance of microscopic clusters and produced consistent turbidity in the cultured medium compared with control. Similar outcomes with addition of bovine lactoferrin hydrolysates (pepsin), bovine transferrin or ovotransferrin suggested that the effect is not lactoferrin-specific. On the other hand, addition of proteins, except bovine transferrin, did not alter surface hydrophobicity. These results indicate that one or more surface components involved in autoaggregation of bifidobacteria are proteins.     

Steric effects of isoleucine 107 on heme reorientation reaction in human myoglobin

Structural factors to regulate the heme reorientation reaction in myoglobin were examined and we found that the side chain at position 107 (Ile107), which is located between the 2-vinyl and 3-methyl groups of heme, forms a kinetic barrier for the heme rotation about the alpha-gamma axis. The phenylalanine-substituted mutant showed an extremely slow heme reorientation rate, compared to that of the wild-type protein, while replacement by the decreased side chain, valine, at position 107 accelerated the reorientation reaction. Considering that the spectroscopic data show only minor structural changes in the heme environments of the Ile107 mutants, the side chain at position 107 sterically interacts with the heme peripheral groups in the activation state for the heme reorientation, which supports the intramolecular mechanism that the heme rotates about the alpha-gamma axis without leaving the "protein cage."     

Temperature sensitization of liposomes by use of N-isopropylacrylamide copolymers with varying transition endotherms

Three kinds of copolymers of N-isopropylacrylamide (NIPAM) with the same conformational transition temperature and varying transition endotherms were synthesized with N-acryloylpyrrolidine (APr), N,N-dimethylacrylamide (DMAM), and N-isopropylmethacrylamide (NIPMAM) as the comonomers. Two dodecyl groups were incorporated into the termini of these copolymers as an anchor for the fixation to a liposomal membrane. Egg yolk phosphatidylcholine liposomes having these copolymers were prepared and their temperature-sensitive contents release and association properties were investigated. While these copolymer exhibited a conformational transition at ca. 40 degrees C, DeltaH for the transition increased in the order of poly(APr-co-NIPAM) < poly(DMAM-co-NIPAM) < poly(NIPMAM-co-NIPAM). The liposomes containing poly(NIPMAM-co-NIPAM) showed a drastic release enhancement of entrapped calcein above the transition temperature, whereas the liposomes with poly(DMAM-co-NIPAM) and those with poly(APr-co-NIPAM) exhibited moderate and slight enhancement of calcein release above that temperature, respectively. On the contrary, the liposomes containing poly(APr-co-NIPAM) showed significant aggregation above the transition temperature, but the aggregation was hardly observed for the liposomes having poly(NIPMAM-co-NIPAM), indicating that poly(APr-co-NIPAM) more efficiently made the liposome surface hydrophobic. Thus, we concluded that the copolymer with a large DeltaH is suitable for obtaining functional liposomes with a temperature-sensitive contents release property, whereas the copolymer with a small DeltaH is appropriate for preparing functional liposomes with a temperature-sensitive surface property.     

Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity

We designed a novel type of cationic lipid, lipids with a cationic polar group in the polyamidoamine dendron, because these dendron-bearing lipids are expected to form complexes with plasmid DNA and achieve efficient transfection of cells by synergy of endosome buffering and membrane fusion with the endosome, both of which are useful for the promotion of the transfer of plasmid DNA from endosome to cytosol. Four kinds of lipids with polyamidoamine dendrons of first to fourth generations, DL-G1, DL-G2, DL-G3, and DL-G4, were synthesized. The lipid with a dendron of a higher generation exhibited greater ability to form lipoplexes with plasmid DNA, as estimated by agarose gel electrophoresis. While the DL-G1 lipoplex did not transfect CV1 cells, the lipoplexes containing the DL-G2, DL-G3, or DL-G4 could induce transfection of the cells, and their activity was elevated with increasing generation of the dendron. Addition of dioleoylphosphatidylethanolamine (DOPE), which is known to increase fusion ability of a lipid membrane, into the lipoplexes greatly enhanced their transfection activity. In addition, the comparison with DC-Chol-containing lipoplex, which is widely used as a nonviral vector, showed that the DL-G3-DOPE lipoplex exhibits more efficient transfections. These findings imply that these dendron-bearing lipids may form the basis for a novel family of cationic lipids for efficient gene delivery.     

Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs

Polyamidoamine dendrimers having poly(ethylene glycol) grafts were designed as a novel drug carrier which possesses an interior for the encapsulation of drugs and a biocompatible surface. Poly(ethylene glycol) monomethyl ether with the average molecular weight of 550 or 2000 was combined to essentially every chain end of the dendrimer of the third or fourth generation via urethane bond. The poly(ethylene glycol)-attached dendrimers encapsulating anticancer drugs, adriamycin and methotrexate, were prepared by extraction with chloroform from mixtures of the poly(ethylene glycol)-attached dendrimers and varying amounts of the drugs. Their ability to encapsulate these drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts. Among the poly(ethylene glycol)-attached dendrimers prepared, the highest ability was achieved by the dendrimer of the fourth generation having the poly(ethylene glycol) grafts with the average molecular weight of 2000, which could retain 6.5 adriamycin molecules or 26 methotrexate molecules/dendrimer molecule. The methotrexate-loaded poly(ethylene glycol)-attached dendrimers released the drug slowly in an aqueous solution of low ionic strength. However, in isotonic solutions, methotrexate and adriamycin were readily released from the poly(ethylene glycol)-attached dendrimers.     

Elevated polyamine levels in erythrocytes of SLC:ICR mice inoculated with ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma cells (4×10⁵ cells/mouse) were inoculated intraperitoneally in 7-week-old SLC:ICR mice, and polyamine levels in peripheral erythrocytes and in ascites cells were determined periodically. Polyamine levels in peripheral erythrocytes increased linearly until 10 days after cell inoculation, while ascites cells showed exponential growth.The effect of carbazilquinone on cellular growth and polyamine levels in erythrocytes was also studied. When 1 or 2mg/kg of carbazilquinone was injected intraperitoneally on day 4 or on day 7, cellular growth was suppressed and the survival time of the mice was lengthened. The polyamine levels in erythrocytes were also markedly decreased 3 days after the carbazilquinone injection.These results suggest that the polyamine levels in peripheral erythrocytes are closely related to the cellular growth of Ehrlich ascites carcinoma cells.     

Casein digestion by Debaryomyces hansenii isolated from cheese

To understand the possible proteolytic contribution of yeast during cheese ripening, Debaryomyces hansenii 212 was isolated from commercial blue-veined cheese and incubated in a medium containing casein. Growth and casein degradation were recognized at the cheese-ripening temperature. Proteolytic activity was found in the intracellular fraction, and the enzyme, which was attached to the cell wall, primarily acted on beta-casein. The cytosol contained more than 90% of the total proteolytic activity which was responsible for the degradation of both alpha(s)- and beta-casein. These results suggest that the contribution of yeast to cheese ripening would depend on the susceptibility to cell lysis in addition to its proteolytic activity.