Enhancement of cell growth by intracellularly expressed herpes simplex virus thymidine kinase

A recombinant cell line (NIH3T3:pLtkSN) was made by infecting parental cells (NIH3T3) with a recombinant retrovirus (pLtkSN) encoding herpes simplex virus thymidine kinase (HSVtk) gene. The cells expressing HSVtk (NIH3T3:pLtkSN) grew 2.3 times more than the parental cells (NIH3T3) in Dulbecco's Modified Eagles Media containing 10% (v/v) horse serum. The NIH3T3:pLtkSN cells also showed a significant enhancement in the maximal cell concentration and the specific growth rate even at 2.5% serum concentration. The specific O2 uptake rate of NIH3T3 was 2.1 times greater than that of NIH3T3:pLtkSN. Under both O2-limited and O2-unlimited conditions, it appears that HSVtk plays an important role in enhancing the growth characteristics of animal cells.     

USE OF BLUE LAKE AS AN INDICATOR OF BACTERIAL PENETRATION INTO EGGS

Blue Lake, an insoluble dye, was evaluated as an indicator of potential bacterial penetration into eggs. Various groups of eggs (fresh-laid; commercial; water-washed) were dipped in 0.25% (w/v) Blue Lake in 0.1% Triton X-100 solution for 2 min and incubated at room temperature up to 1 ± 24 h. Penetration was detected by counting the blue dots on shell membranes after breaking the eggs. Commercial eggs allowed the easiest penetration. All commercial eggs showed blue dots even at 2 min incubation and the average count reached 111/egg at 1 h. Water-washed eggs allowed much less penetration than commercial eggs and the counts of blue dots on those eggs were 10/egg at 2 min and 22 at 1 h. Fresh-laid eggs did not allow any penetration up to 24 h. Above results corresponded very well with the penetration study with Salmonella enteritidis and also with the morphological study of eggshell surfaces using electron microscopy where fresh-laid eggs had intact cuticle layers, but commercial eggs did not. Thus, Blue Lake dye might be used as a rapid indicator of bacterial penetration through eggshells.     

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.     

Lipid Metabolism in Fucus serratus as Modified by Environmental Factors

The effect of changes in the environment on lipid metabolismhas been studied in the brown alga, Fucus serratus L. Lightstimulated the incorporation of radioactivity from /{^I4C/}acetateinto oleic and, especially, into linoleic acid. The same effectwas caused by lowering the incubation temperature from 15 °Cto 4 °C. Incubations in the presence of Cd ^{+ +}, Pb ^{+ +} orZn^{+ +} had no effect on the total uptake of /{¹⁴C/}acetate intothe frond tip samples, but lowered the labelling of total lipidsrelative to aqueous-soluble components. However, pre-exposureof the algae to heavy metal cations caused changes in the uptakeof radioactivity but had less effect on the relative labellingof lipids than incubations in the presence of heavy metal cations.Algae collected from sites where the dissolved levels of heavymetals were elevated, showed a decrease in the relative labellingof lipids from /{¹⁴C/}acetate. Concentrations of Cd^{+ +}, Pb⁺⁺ or Zn^{+ +} at 10 x levels found at the collection site had littleeffect on the pattern of fatty acids made by Fucus serratus. Key words: Lipid metabolism, Fucus serratus L., Environmental changes     

The Nuclear Envelope of Amoeba proteus During Mitosis as Studied With a Monoclonal Antibody Against a Membrane-Associated Protein

. The behavior of nuclear envelopes during mitosis in Amoeba proteus was studied by means of indirect immunofluo-rescence staining using a monoclonal antibody against a 220-kD membrane-associated protein of amoebae in conjunction with DAPI staining of chromatin. The antibody selectively recognized antigens on nuclear envelopes during interphase but did not react with the nuclear membranes during mitosis until after cytokinesis had been completed. Thus, it appeared that the membrane-associated protein reacting with the monoclonal antibody and normally present on the nuclear membranes was absent from fragmented nuclear membranes or nuclear membranes that were continuous but did not have the honey-comb lamina. The findings suggested that the 220-kD nuclear-membrane protein may be involved in the dissolution and reformation of the honey-comb lamina during mitosis in amoebae.     

Immune-Enhancing Activity Screening on Extracts from Two Crickets, Gryllus bimaculatus and Teleogryllus emma

This study was performed to explore novel and valuable uses of insect resources, important subjects of the natural compound used in bio‐industries. The whole bodies of two crickets, Gryllus bimaculatus and Teleogryllus emma, selected from medicinal insect species, were carefully ground and treated with 80% EtOH. The insect extracts were solubilized and separated by hexane, butanol, and D.W according to their polarities. Three types of extracts, a D.W fraction (G1) and a boiling extract (G2) of an introduced cricket, G. bimaculatus, and a D.W fraction (T1) of a Korean local cricket, T. emma, were prepared to assay immune stimulating activity of cricket originated compounds. The all of three treated cricket extracts showed to increase IL‐4, IFN‐, and TNF‐α. Among those extract, extract G2, boiled extract from G. bimaculatus, was the best immune–enhancing fraction. The results of this study could be fundamental information for further works to use insects as natural resources having plenty of potentials and varieties.     

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.