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Cholesterol is an important cell membrane component and has been used as co-lipid for cationic liposome to enhance gene delivery. However, the role of cholesterol in transfection efficiency has not been fully understood. In this study, transfection efficiency of liposome was measured after cholesterol was added to the cell culture medium. As a result, addition of cholesterol increased transfection efficiency of several liposomes consisting of different lipid components in various cells (AGS, CHO, COS7 and, MCF7). Furthermore, treatment of cells with cholesterol modulating drugs, imipramine and U18666A, also increased transfection efficiency of liposomes. To elucidate the role of added cholesterol in gene transfer, endocytotic mechanism was studied and also revealed that adding cholesterol in culture media induced participation of caveolae-mediated endocytosis and micropinocytosis in CHO cell. Therefore, the results of this work suggest that modulation of intracellular cholesterol can be an important method to enhance gene delivery.

Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) differ markedly in their inhibition by GTP. These regulatory preferences must arise from amino acid residues that are not common between hGDH isozymes. We have constructed chimeric enzymes by reciprocally switching the corresponding amino acid segments 390–465 in hGDH isozymes that are located within or near the C-terminal 48-residue antenna helix, which is thought to be part of the regulatory domain of mammalian GDHs. These resulted in triple mutations in amino acid sequences at 415, 443, and 456 sites that are not common between hGDH1 and hGDH2. The chimeric enzymes did not change their enzyme efficiency ( k cat / K m ) and expression level. Functional analyses, however, revealed that the chimeric mutants almost completely acquired the different GTP regulatory preference between hGDH isozymes. These results suggest that the 415, 443, and 456 residues acting in concert are responsible for the GTP inhibitory properties of hGDH isozymes.

Ethyl formate (EF) was tested in mixtures with natural products (NPs) as a fumigant against the internal stages of Sitophilus oryzae. These novel formulations of EF [EF:NPs = 9.0:1.0, (v/v)] were shown to be chemically stable for 1 month after initial mixing. Therefore, EF acts as a good solvent for various NPs having insecticidal activity. The toxicity of EF alone, and of the various EF formulations, was evaluated on each developmental stage at different concentrations and exposure times. The results show that the effect of EF alone, as compared with the various EF formulations, was similar or lower in the mixed age cultures (MAC) of S. oryzae after 6 hr of exposure. For the pupal stage of S. oryzae, EF + thujone showed increased toxicity (>20%) at both 6 hr of exposure at 67.4 mg/L, as well as at 24 hr of exposure at 37.6 mg/L. Also, EF + thujone, EF + menthone, and EF + carvone showed higher toxicities as compared to EF alone, on the pupal stage of S. oryzae after 24 hr of exposure at 37.6 mg/L. However, we couldn't find significant differences in the formulations in terms of their synergistic effects. The most significant result of this research was the ability to use EF as a solvent for the application of various natural materials as fumigants and/or protectants. Our continuing research is aimed at finding natural product formulations that possess enhanced toxicity with respect to the internal stages of stored grain pests, as well as low mammalian toxicity.

Fish materials were tested for their attractiveness to the bean bug, Riptortus clavatus (Thun-berg) (Heteroptera: Alydidae) in a soybean field. Fish materials' volatiles evoked male-specific responses in attractiveness. Fish materials such as mackerel, hairtail, and gizzard shad attracted significantly higher number of males than control, but they were not attractive to females. The attractiveness of MeOH extract of mackerel to R. clavatus males was not different from 10 live males and aggregation phe-romone traps of R. clavatus. Further studies are needed to identify volatile compound(s) from fish smell responsible for the attraction of R. clavatus males.

Rats were fed 100 μM aluminum maltolate for one year in their drinking water. Brain aluminum contents have increased 4.2-fold in the aluminum-treated group, whereas no significant changes in the body weight, brain weight, and brain protein content were observed. Long-term aluminum feeding induced apoptosis as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and showed activatory effects on the catalytic efficiency ( k cat / K M ) of monoamine oxidase-A and monoamine oxidase-B up to 1.9- and 3.8-fold, respectively. The expression level of monoamine oxidase isotypes on the Western blot remained unchanged between the two groups, suggesting a change in post-translational regulation of the activities of monoamine oxidase isotypes by long-term aluminum feeding.

When intracisternally injected to rat brain, aluminum induced apoptosis as assessed by DNA fragmentation and activation of caspase-3 and caspase-12. Co-administration of glial cell line-derived neurotrophic factor (GDNF) effectively prevented aluminum-induced cell death through reduced apoptosis whereas brain-derived neurotrophic factor (BDNF) accelerated aluminum-induced apoptosis, suggesting that the extent of aluminum neurotoxicity in vivo may depend on the biological activity of the neurotrophic factors.

In the present study, the cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the human glutamate dehydrogenase isozymes (hGDH1 and hGDH2). None of the mutations tested affected the expression or stability of the proteins. There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The K(M) values for glutamate were 4-10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2, whereas no differences in the K(M) values for NAD(+) were detected between the mutant and wild-type enzymes. For K130Y mutant, the K(M) value for glutamate increased 1.6-fold, whereas the catalytic efficiency (k(cat)/K(M)) showed only 2-3% of the wild-type. Therefore, the decreased catalytic efficiency of the K130 mutant mainly results from the reduced k(cat) value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The D172Y mutant did not show any changes in k(cat) value and K(M) values for glutamate and NAD(+), indicating that D172Y is not directly involved in catalysis and substrates binding of the hGDH isozymes. For sensitivity to ADP activation, only the D172Y mutant showed a reduced sensitivity to ADP activation. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. There were no differences in their sensitivities to GTP inhibition between the wild-type and mutant GDHs at all positions tested. Our results suggest that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.