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.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

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.     

A New Species of Poorly Known Mayfly Genus Paegniodes Eaton (Ephemeroptera: Heptageniidae) from Vietnam

A heptageniid mayfly species Paegniodes dao sp. nov. (Ephemeroptera: Heptageniidae) is described from Vietnam. The larva is distinguished by round anterolateral margins of pronotum, triangular‐shaped labrum, rudimentary lamella and well developed fibrillae of gills 1, and light brown body that lacks distinct markings. Diagnosis, line drawings, material, and habitat data are provided.     

A survey for Cyclospora spp. in Kenyan primates, with some notes on its biology.

From March 1999 through August 2000, 511 stool samples collected from 11 different primate species in 10 geographically distinct locations in Kenya, East Africa, were screened for the presence of Cyclospora spp. oocysts. Positive samples (43/102, 42%) were identified in vervet monkeys (Cercopithecus aethiops) in 4 of 4 locations; 19/206 (9%) in yellow and olive baboons (Papio cynocephalus, P. anubis, respectively) in 5 of 5 locations; and 19/76 (25%) in black and white colobus monkeys (Colobus angolensis, C. guereza, respectively) from 2 of 3 locations. DNA sequences obtained from 18 S rRNA coding regions from respective subsets of these positive samples were typed as Cyclospora cercopitheci (samples from Cercopithecus aethiops). Cyclospora papionis (samples from Papio cynocephalus and P. anubis), and Cyclospora colobi (samples from Colobus angolensis and C. guereza). Cyclospora oocysts were not detected in samples collected from patas, highland sykes, lowland sykes, blue sykes, DeBrazza, or red-tailed monkeys. A coded map showing the geographic location of the collected samples is given. Stool samples from 1 troop of vervet monkeys were collected over a 12-mo period. Positive samples ranged between 21 and 63%. These results suggest that there is no strongly marked seasonality evident in Cyclospora infection in monkeys as has been noted in human infection. This is further confirmed by the recovery of positive samples collected from vervet monkeys, baboons, and colobus monkeys at all times of the year during this survey. This absence of seasonality in infection is especially notable because of the extreme weather patterns typical of Kenya, where marked rainy and dry seasons occur. A second noteworthy observation is that the striking host specificity of the Cyclospora species initially described was confirmed in this survey. Baboons were only infected with C. papionis, vervet monkeys with C. cercopitheci, and colobus monkeys with C. colobi, despite geographic overlaps of both the monkey and parasite species and wide geographic distribution of each parasite and monkey host.     

Exciton interactions in synthetic porphyrin dimers

The Soret absorption spectra of six synthetic rigid porphyrin dimers whose crystal structures have been determined are simulated using simple exciton theory. The objective is to test the validity of the point dipole and associated approximations; the electronic interaction parameters are thus calculated using data obtained from the monomer spectra, with no adjustable parameters. Satisfactory agreement between theory and experiment is obtained for one class of dimers but not for a second. This poses a challenge for semiempirical electronic structure methods as to whether improvements over the point dipole calculations can be obtained.     

N-linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H-ras were analyzed by 500-MHz 1H-NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D-[3H]glucosamine or L-[3H]fucose and the glycopeptides included in Bio-Gel P-10 (Mr 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues. The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by 1H-NMR spectroscopy to be tetraantennary containing terminal Gal alpha 1----3. (formula; see text) This structure was verified by binding to the immobilized alpha-Gal-specific lectin, Griffonia simplicifolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain tri- or tetraantennary glycopeptides. In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAc alpha 2----3 in addition to Gal alpha 1----3. (formula; see text) In attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAc alpha 2----3 reduced the affinity to L-PHA - agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAc alpha 2----3 but no NeuNAc alpha 2----6 or Gal alpha 1----3. A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNAc alpha 2----3, the presence of NeuNAc alpha 2----6 and Gal alpha 1----3. On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri- or tetraantennae must be sialylated in alpha 2----3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in alpha-Gal residues, whereas after transformation alpha-Gal residues appear to be replaced by NeuNAc alpha 2----3.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of protein kinases and protein phosphatases in the regulation of cardiac sarcoplasmic reticulum function

Summary Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3,5-monophosphate (cAMP)-dependent and by calcium · calmodulin-dependent protein kinases on a 27 000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca²⁺ transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the CAMP-dependent and the calcium · calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.     

Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Chromosome aberrations in workers of nuclear-power plants

The frequencies of chromosome aberrations in 135 workers from nuclear-power plants were compared with those in 135 age-matched controls. A total of 135,000 cells was scored. The frequencies of dicentric chromosome were 1.67 × 10⁻³ in the exposed group and 0.49 × 10⁻³ in the control group and those of chromosome-type deletion were 3.33 × 10⁻³ and 1.10 × 10⁻³, respectively. The frequencies of all types of chromosome aberrations in the exposed subjects were higher than those in the control group, but no significant trend of dose-dependent increase was observed when only the exposed group were considered. Poisson regression analysis, with both exposed and control included, showed that there was a significant association of chromosome aberration with radiation dose and the duration of work, but not with age, smoking habit and alcohol intake. It was also found that recent exposure to radiation, within the last 5 years, had contributed more to the observed chromosome aberration than earlier exposure.