TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells

Human glutamate dehydrogenase (GDH) gene was fused with a gene fragment encoding the nine amino acid (RKKRRQRRR) protein transduction domain of human immunodeficiency virus TAT protein in bacterial expression vector to produce genetic in-frame TAT-GDH fusion protein. The TAT-GDH protein can enter PC12 cells efficiently when added exogenously in culture media as determined by Western blot analysis and enzyme activities. Once inside the cells, the transduced denatured TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence. TAT-GDH fusion protein and TAT itself showed no cytotoxicity in PC12 cells. Although the exact mechanism of transduction across a membrane remains unclear, the transduction activity of TAT-GDH into PC12 cells may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.     

Controlled activity of mouse astrocytes on electrospun PCL nanofiber containing polysaccharides from brown seaweed

The central nervous system (CNS), once injured, rarely recovers original function mainly due to its limited regeneration ability. Astrocytes are cells that play critical roles in neural regeneration. Several biomaterials have been studied to replace and regenerate lost tissues within injured CNS. Seaweeds have extracellular polymeric substances (EPS) with bioactive properties such as antiviral and antioxidant properties. In this study, astrocyte activity was assessed, after being cultured on an electrospun polycaprolactone (PCL) nanofibrous mat containing a brown seaweed EPS. Laminarin and fucoidan, two main components of EPS extract from the brown seaweed, were concluded to increase or decrease astrocyte activity with respect to their concentration. When the concentration was under 10 ??g/ml, the astrocytes tended to increase their viability. In contrast, over 10 ??g/ml EPS in media suppressed the viability of astrocytes. In addition, when contained in PCL nanofiber, the EPS extract was also proven to influence astrocyte activity in the same way as the case when astrocytes were exposed to EPS in solution. This implies that the brown seaweed EPS?CPCL nanofiber mat can be used for temporal control of astrocyte activity by EPS concentration. Through this research, we propose that the electrospun EPS?CPCL nanofiber could be used as a nanomedicine or scaffold to treat CNS injuries.     

Medicinal herb extracts inhibit growth of<Emphasis Type="Italic">Rhabditis elongate</Emphasis> (Nematoda:<Emphasis Type="Italic">Rhabditidae</Emphasis>)

We tested the influence of extracts from three medicinal herbs —Salvia miltiorrhiza, Schizandra chinensis, andEugenia caryophyllata — on activity of the nematodeRhabditis elongate. Treatment with f.caryophyllata was most useful, causing the greatest decrease in populations and mobility, but did not have any detrimental effect on the initial growth of the host microorganism,Escherichia coli. For example, when 0.5 g/L of the extract was added to an inoculated liquid culture, we counted 710 nematodes/mL, with a multiplication rate 5 times greater than the initial population. This was in contrast to the control sample, which had a count of 1100 nematodes/mL and a growth ratio of 11. For our field test, nematode mobility in the presence of the extract also decreased, to 6.8 mm/day, compared with 20 mm/day for the control. Likewise, when 1.0 g/L of the extract was added to the soil, the total number of nematodes was reduced to only 30- to 40% of the control population.     

Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.     

SIRT1 and c-Myc Promote Liver Tumor Cell Survival and Predict Poor Survival of Human Hepatocellular Carcinomas

The increased expression of SIRT1 has recently been identified in numerous human tumors and a possible correlation with c-Myc oncogene has been proposed. However, it remains unclear whether SIRT1 functions as an oncogene or tumor suppressor. We sought to elucidate the role of SIRT1 in liver cancer under the influence of c-Myc and to determine the prognostic significance of SIRT1 and c-Myc expression in human hepatocellular carcinoma. The effect of either over-expression or knock down of SIRT1 on cell proliferation and survival was evaluated in both mouse and human liver cancer cells. Nicotinamide, an inhibitor of SIRT1, was also evaluated for its effects on liver tumorigenesis. The prognostic significance of the immunohistochemical detection of SIRT1 and c-Myc was evaluated in 154 hepatocellular carcinoma patients. SIRT1 and c-Myc regulate each other via a positive feedback loop and act synergistically to promote hepatocellular proliferation in both mice and human liver tumor cells. Tumor growth was significantly inhibited by nicotinamide in vivo and in vitro. In human hepatocellular carcinoma, SIRT1 expression positively correlated with c-Myc, Ki67 and p53 expression, as well as high á-fetoprotein level. Moreover, the expression of SIRT1, c-Myc and p53 were independent prognostic indicators of hepatocellular carcinoma. In conclusion, this study demonstrates that SIRT1 expression supports liver tumorigenesis and is closely correlated with oncogenic c-MYC expression. In addition, both SIRT1 and c-Myc may be useful prognostic indicators of hepatocellular carcinoma and SIRT1 targeted therapy may be beneficial in the treatment of hepatocellular carcinoma.     

Cycloheximide resistance in carrot culture: a differentiated function

Cultured carrot cells grow as unorganized callus tissue in medium containing auxin. Upon removal of the auxin from the medium, they grow in an organized manner and differentiate into embryos. In the normal cell line, W001C, the callus growth can be inhibited by cycloheximide, but the embryonic growth cannot. A variant cell line, WCH105, whose callus growth is resistant to cycloheximide, was isolated. The mechanism of cycloheximide resistance in embryos of both lines and in WCH105 callus was found to be cycloheximide inactivation. In addition to auxin, bromodeoxyuridine can also promote callus growth in carrot culture. Callus cultures maintained by bromodeoxyuridine behave the same as do those maintained by auxin. WCH105 callus is resistant, whereas W001C callus is sensitive to cycloheximide inhibition. Except for the onset of embryogenesis, cycloheximide inactivation is expressed throughout the embryo developmental stages up to the plantlets. These results suggest that cycloheximide inactivation is a function expressed in the differentiated, but not in the undifferentiated, tissues.     

Cytological Studies on the Embryogenesis of Hybrids between Oryza sativa L.(Pennisetum sp.

(1) The pollen grains of Pennisetum can germinate normally on the stigma of rice and the pollen tubes can grow into the style and enter the embryo sacs. However, the process of double fertilization is slow and more or less abnormal and phenomenon of simple fertilization often occurs. (2) It has been found that in the majority of cases the development of the embryos is slow and stays long in the stage of globular embryos, thus, the differentiation of the embryos is very difficult and degeneration of the embryos appears many times. Simple differentiation was observed only in some embryos during 16–24 days after pollination. Normal differenting and developing embryos were not observed. The cause of the degeneration of the embryos is related to the state of endosperm development and also to the non-coordination of the genomes of both parents. (3) The development of the endosperm is abnormal. The change from the free nuclei into the cells in the endosperm is delayed as late as the 8th day after pollination. The whole endosperm tissue is composed of the cell masses which are quite different both in shape and function, a part of these endospemn cells lacks the ability to synthesize starch. The disintegration of the endosperm could be frequently observed during their development. (4) A lots of starch are accumulated in the nucellar cells near the antipodals, It is shown that there was some metabolic confusion resulted from the crossing in the embryo sacs. Based on the above mentioued results the authers consider that the failure of producing seeds by crossing is at least related to the nutrient condition which are essential for the development of embryos. If embryo culture technique is employed at the early stage of the embryo development the hybrid seeds could be obtained.     

Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.