Alkaline Phosphatase Content and the Effects of Prednisolone on Mammalian Cells in Culture

The alkaline phosphatase content of different tissue culture cell lines has been shown to vary from no detectable activity to high enzyme concentration. Within the epithelial lines studied alkaline phosphatase is either constitutive or inducible. Two epithelial cell strains in which alkaline phosphatase was "absent" could be induced to develop significant amounts of the enzyme when grown in the presence of Δ¹-hydrocortisone. Phosphate did not repress enzyme induction by prednisolone. Under conditions of deadaptation the induced enzyme was diluted by cell multiplication. The mouse fibroblastic L line and several human fibroblastic lines did not contain alkaline phosphatase when grown under the conditions described nor could they be induced to produce the enzyme when cultivated in medium with prednisolone. Δ¹-Hydrocortisone has other characteristic effects on established mammalian cell cultures which vary among cell lines. Human epithelial lines show reduction in cell multiplication with increase in mitotic index. The cytoplasm is increased and cell volume is nearly doubled. Mouse fibroblasts show a similar reduction in cell multiplication with a decrease in mitotic index. There is no increase in cell cytoplasm. Human fibroblast strains show no inhibition of multiplication or alteration in total cell protein when grown in medium containing prednisolone. Antisera prepared against "negative" prednisolone-inducible human cell lines and against a positive human line inhibited alkaline phosphatase activity to an equal degree.     

Charged-particle mutagenesis. 1. Cytotoxic and mutagenic effects of high-LET charged iron particles on human skin fibroblasts.

Cytotoxic and mutagenic effects of high-LET charged iron (56Fe) particles were measured quantitatively using primary cultures of human skin fibroblasts. Argon and lanthanum particles and gamma rays were used in comparative studies. The span of LETs selected was from 150 keV/microns (330 MeV/u) to 920 keV/microns (600 MeV/u). Mutations were scored at the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus using 6-thio-guanine (6-TG) for selection. Exposure to these high-LET charged particles resulted in exponential survival curves. Mutation induction, however, was fitted by the linear model. The relative biological effectiveness (RBE) for cell killing ranged from 3.7 to 1.3, while that for mutation induction ranged from 5.7 to 0.5. Both the RBE for cell killing and the RBE for mutagenesis decreased with increasing LET over the range of 1.50 to 920 keV/microns. The inactivation cross section (sigma i) and the action cross section for mutation induction (sigma m) ranged from 32.9 to 92.0 microns2 and 1.45 to 5.56 X 10(-3) microns2; the maximum values were obtained by 56Fe with an LET of 200 keV/microns. The mutagenicity (sigma m/sigma i) ranged from 2.05 to 7.99 X 10(-5) with an inverse relationship to LET.     

Ventilatory and hematopoietic responses to chronic hypoxia in two rat strains.

Hilltop (H) and Madison (M) strains of Sprague-Dawley rats exhibit strikingly different susceptibilities to the effects of chronic altitude exposure. The H rats develop greater polycythemia, hypoxemia, and pulmonary hypertension. We studied ventilation, pulmonary gas exchange, tissue oxygenation, and hematologic adaptations in the two rat strains during a 50-day exposure to a simulated altitude (HA) of 5,500 m (18,000 ft). There were no strain differences among the variables we studied under sea level (SL) conditions. Within the first 14 days of hypoxic exposure, the only significant strain differences were that erythropoietin (EPO) rose much higher and erythroid activity was greater in the H rats, even though arterial Po2 and PCo2 (Pao2 and PaCo2, respectively), renal venous PO2 (Prvo2), and ventilation (VE) were equivalent in the two strains during this time. By day 14 at HA, the H rats had significantly higher erythroid activity, hematocrit (Hct), and EPO levels, significantly lower PaO2 and PrvO2, but equivalent VE and PaCO2. These changes persisted for the remainder of the exposure, except that the Hct continued to rise and the increase was greater in H rats. Despite the greater O2-carrying capacity of H rats in the later stages of hypoxic exposure, PaO2 and PrvO2 were significantly lower in H rats. There were no strain differences at either SL or HA in ventilatory responses to hypercapnia or hypoxia, in blood O2 affinity or 2,3-diphosphoglycerate, in extrarenal production of EPO, or in EPO clearance. We conclude that early in the hypoxic exposure the H rats produce more EPO at apparently equivalent levels of hypoxia, and this is the first step in the pathogenesis of the maladaptation to HA manifest by H rats. We find no consistent evidence that differences in VE contribute to the variable susceptibility to hypoxia in the two rat strains.     

GLOBAL POPULATION STRUCTURE AND NATURAL HISTORY OF THE GREEN TURTLE (CHELONIA MYDAS) IN TERMS OF MATRIARCHAL PHYLOGENY

To address aspects of the evolution and natural history of green turtles, we assayed mitochondrial (mt) DNA genotypes from 226 specimens representing 15 major rookeries around the world. Phylogenetic analyses of these data revealed (1) a comparatively low level of mtDNA variability and a slow mtDNA evolutionary rate (relative to estimates for many other vertebrates); (2) a fundamental phylogenetic split distinguishing all green turtles in the Atlantic-Mediterranean from those in the Indian-Pacific Oceans; (3) no evidence for matrilineal distinctiveness of a commonly recognized taxonomic form in the East Pacific (the black turtle C.m. agassizi or C. agassizi); (4) in opposition to published hypotheses, a recent origin for the Ascension Island rookery, and its close genetic relationship to a geographically proximate rookery in Brazil; and (5) a geographic population substructure within each ocean basin (typically involving fixed or nearly fixed genotypic differences between nesting populations) that suggests a strong propensity for natal homing by females. Overall, the global matriarchal phylogeny of Chelonia mydas appears to have been shaped by both geography (ocean basin separations) and behavior (natal homing on regional or rookery-specific scales). The shallow evolutionary population structure within ocean basins likely results from demographic turnover (extinction and colonization) of rookeries over time frames that are short by evolutionary standards but long by ecological standards.     

Screening and analysis of differentially expressed genes from an alien addition line of wheat Thinopyrum intermedium induced by barley yellow dwarf virus infection.

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.     

THE DISCOVERY OF THE GENUS SHELFORDIA CAMERON (HYMENOPTERA: BRACONTOAE: BRACONINAE) IN CHINA, WITH DESCRIPTION OF ONE NEW SPECIES

The genus Shelfordia is discovered in China for the first time, and one new species of this genus (S. chinensis sp. nov.) is fully described and illustrated in the present paper. The type specimen is deposited in the Parasitic Hymenoptera Collection, Zhejiang University, Hangzhou, China.     

EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

USE OF A PETHIDINE AND MIDAZOLAM COMBINATION FOR THE REVERSIBLE SEDATION OF CRABEATER SEALS (LOBODON CARCINOPHAGUS)

Between October and December of 1996–1999, off eastern Antarctica (60°-150°E), we darted 31 crabeater seals with midazolam and pethidine at estimated dose rates of 0.15–0.4 mg/kg and 1–3 mg/kg, respectively. Maximum sedation was reached at 23 ± 9 min (n = 18) and first signs of recovery were noted at 54 ± 24 min (n = 4). Seals greater than 250 kg body-mass were sedated by administration of approximately 90–100 mg midazolam and 600 mg pethidine, but the degree of sedation was unpredictable and did not permit invasive procedures in some cases. Behavior of the seal and adjacent conspecifics affected the success of procedures and our ability to monitor vital signs. Naloxone and flumazenil reversed sedation, making this combination attractive for use in animals adjacent to water. Additional ketamine was administered to two seals, resulting in improved restraint.