Irradiation of cells in tissue culture

Summary Cultures of human amnion were employed to check the hypothesis that cell strains with heteroploid chromosome counts regularly produce giant cells within 12 days following treatment with 2000 r and 4000 r of gamma irradiation from a cobalt source, while this response has not been obtained from primary cultures whose cells were presumed to be diploid.The giant cell reaction not only was obtained from two transfer passage lines of a well-established amnion strain developed at Berkeley (No A 185-21C-26 and No A 185-21C-45) but was also found for a 20-day second passage culture of amnion. Since this line has continued to reproduce at a rapid rate, it is presumed to have assumed the features of a typical strain within the period of observation. This impression was reinforced by the finding that the chromosome number for 32 cells fixed on the 35th day had a modal value of 67.In contrast, both untrypsinized and trypsinized spindle cells in primary cultures as well as unaltered epithelial elements which had not been subcultured gave no evidence of giant cell formation 12 days after exposure to 2000 r and 4000 r from a Cobalt⁶⁰ source.These data lend evidence that giant cell formation is related to the chromosomal constitution of the irradiated elements.This research was supported by funds provided under Contract AF 18(600)-1263 with the School of Aviation Medicine, USAF, Randolph Air Force Base, Texas.Fellow of the Instituto de Alta Cultura and the Fundacão Calouste Gulbenkian of Lisbon, Portugal.Tobacco Industry Research Committee Fellow.     

A first-generation map of the turkey genome.

A primary linkage map of the domestic turkey (Meleagris gallopavo) was developed by segregation analysis of genetic markers within a backcross family. This reference family includes 84 offspring from one F1 sire mated to two dams. Genomic DNA was digested using one of five restriction enzymes, and restriction fragment length polymorphisms were detected on Southern blots using probes prepared from 135 random clones isolated from a whole-embryo cDNA library. DNA sequence was subsequently determined for 114 of these cDNA clones. Sequence comparisons were done using BLAST searches of the GenBank database, and redundant sequences were eliminated. High similarity was found between 23% of the turkey sequences and mRNA sequences reported for the chicken. The current map, based on expressed genes, includes 138 loci, encompassing 113 loci arranged into 22 linkage groups and an additional 25 loci that remain unlinked. The average distance between linked markers is 6 cM and the longest linkage group (17 loci) measures 131 cM. The total map distance contained within linkage groups is 651 cM. The present map provides an important framework for future genome mapping in the turkey.     

Breeding success and chronology of Wood Storks Mycteria americana in northern and central Florida, U.S.A.

The breeding success and chronology of Wood Storks Mycteria americana were studied at eight colonies in northern and central Florida during 1981–1985. Mean ± s.d. clutch size for all colony-years was 3.07 ± 0.56 (n = 2694 nests), with three-egg clutches (72%) most frequent. Mean clutch size among all colonies and years ranged from 2.73 ± 0.55 to 3.41 ± 0.61. Many colonies exhibited significant negative trends in clutch size with, hatching date because of a proportional decrease in four-egg clutches later in the season. Mean colony clutch size was not correlated with nest numbers, nesting density or mean hatching date within most years. Mean ± s.d. number of fledglings for all colonies and years was 1.29 ± 1.16 fledglings per nest (n = 2812 nests). Mean annual fledging rates in colonies ranged from 0 (colony failed) to 2.66 fledglings per nest. Most breeding failure occurred prior to egg hatching, and the second highest mortality occurred between hatching and 2 weeks of age. Four-egg clutches fledged more storks than three-egg clutches, which in turn were more successful than two-egg clutches. However, all clutch sizes showed similar fledgling per egg rates. The seasonal decline in productivity was associated proportionally with smaller clutch sizes later in the breeding season. An increase in mean hatching date was correlated with an increase in latitude. There was greater within-year breeding synchrony among colonies than interyear breeding synchrony within each colony. Breeding synchrony was not correlated with mean hatching date, latitude, longitude, nest numbers or nesting density.     

Molecular analysis of the feline immunodeficiency virus protease: generation of a novel form of the protease by autoproteolysis and construction of cleavage-resistant proteases.

The feline immunodeficiency virus (FIV) protease is essential for virion maturation and subsequent viral replication in that it cleaves the Gag and Gag/Pol polyproteins at eight sites to release the respective structural proteins and enzymes. During purification of a recombinant FIV protease (PR), we noted that it underwent autoproteolysis (autolysis) to give discrete cleavage products. These additional PR cleavage sites were defined using N-terminal amino acid sequence analysis and mass spectrometry. Protease breakdown products were also found in FIV virions and were of the same apparent molecular weights as the in vitro autolysis products. Four primary PR autolysis sites were blocked via substitution of either the P1 amino acid with a beta-branched amino acid or the P1' amino acid with lysine. Cleavage-resistant PRs which had Km and k(cat) values similar to those of FIV PR were constructed. An autolysis time course determined that blocking all four primary autolysis sites yielded a cleavage-resistant PR which was enzymatically stable. Concomitant with autolysis is the generation of an N-terminally truncated form of the PR (Thr6/PR) which has enhanced stability with respect to that of FIV PR. A structural basis for the Thr6/PR activity is presented, as are the possible roles of autolysis in the viral replication cycle.     

T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.     

A New Malaria Parasite Plasmodium (Sauramoeba) heischi in Skinks (Mabuya striata) from Nairobi, with a Brief Discussion of the Distribution of Malaria Parasites in the Family Scincidae

ABSTRACT. A new species of malaria parasite, Plasmodium (Sauramoeba) heischi , is described from African skinks (Mabuya striata). Eleven individuals of 90 specimens collected in Nairobi were found to be infected. The new parasite is a large species, characterized by spindle-shaped gametocytes, the female often with a subterminal nucleus. The schizonts produce up to 65 nuclei and cause great hypertrophy and distortion of the host cell. Although similar to P. (Sauramoeba) giganteum in dimensions and merozoite numbers, P. heischi is easily distinguished by gametocyte and schizont shapes.     

Pollen frequencies reflect vegetation patterns in a great basin (U.S.A.) mountain range

Five indices are used to quantify the relationship between vegetation and pollen in a mountain range in the arid Great Basin. Computations are based on vegetation coverage and pollen percentages from 63 stands. Association is a measure of whether the presence of the pollen type in a surface sample is an indication of the presence of the parent plant in the local vegetation. Over-representation and under-representation measure tendencies for pollen to occur where the parent plants are absent and vice versa. The correlation coefficient measures the relationship between plant and pollen in stands where both are present. Twenty-nine trees, shrubs, and herbs accurately reflect local vegetation conditions. A percentage diagram shows elevational trends in abundant pollen types. Regional pollen types are used to compute the accumulation rate of pollen in the surface samples. A diagram of pollen accumulation rates shows trends similar to those shown in the percentage diagram. The moss polsters used in this study may collect pollen over a fifteen-year interval.     

The Spontaneous Formation of Antheridia in Onoclea is not Blocked by Light

Onoclea sensibilis gametophytes were grown from spores on ashedsoil and agar to determine if the spontaneous formation of antheridiacan be blocked by light. Under most conditions, dark-grown gametophytesformed antheridia later than or at the same time as gametophytesgrown in the light. Under no circumstances was there a rapidonset of maleness in the dark. These results contradict thehypothesis that, in Onoclea, antheridiogen is required to inducemaleness because light inhibits the formation of antheridia.In the light, antheridia formed on heart-shaped thalli. In darkness,antheridia formed on filamentous gametophytes. The timing ofonset of maleness was affected by temperature and the presenceof sucrose. The effect of sucrose on the comparison betweenlight and dark treatments depended on both substrate and temperature Onoclea sensibilis, L., sensitive fern, fern gametophytes, sexuality, light-induced block     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.