Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.     

Diaphragm fatigue induced by inspiratory resistive loading in spontaneously breathing rabbits

Diaphragmatic contractility was assessed in spontaneously breathing ketamine-anesthetized rabbits by measuring the strength of diaphragmatic contraction in response to bilateral supramaximal phrenic nerve stimulation at frequencies between 10 and 100 Hz. During 10-180 min of inspiratory resistive loading, contractility decreased by approximately 40%, and hypoxemia and both respiratory and lactic acidosis developed. After 10 min of recovery, both the response to high-frequency stimulation (100 Hz) and the arterial PO2 and PCO2 returned to base-line levels, whereas metabolic acidosis and reduced response to low-frequency stimulation (10-20 Hz) persisted. Similar levels of hypoxemia and respiratory acidosis in the absence of inspiratory resistive loading did not alter diaphragmatic contractility. We conclude that in anesthetized rabbits excessive inspiratory resistive loading results in partially reversible diaphragm fatigue of the high- and low-frequency types, accompanied by hypoventilation and lactic acidosis.     

Membrane systems of freeze-etched striated muscle

The organization of flight muscle of a dragonfly, with special reference to the fibre membrane systems, has been investigated by means of platinum-carbon replicas of freeze-etched material. The fractured surface plasma membrane and origin of transverse tubules are described together with the radial course of the tubules into the cell, along grooves in the mitochondrial surface. The form of the sarcoplasmic reti, culum cisternae is also described. Replicas are interpreted as revealing up to three unfractured membrane surfaces and several fractured profiles. The illustrations include stereo-micrographs of replicas.     

Amino acids as determinants of host preference for the xylem feeding leafhopper,Homalodisca coagulata (Homoptera: Cicadellidae)

Summary Homalodisca coagulata is a highly polyphagous xylem feeder with distinct seasonal patterns in it's selection of host plants. These patterns were examined in relation to the amino acid content of the xylem for four common host species; Lagerstroemia indica, Baccharis halimifolia, Prunus persica, and Prunus salicina. Xylem fluid was collected from each host species at times when numbers of feeding leafhoppers were both low and high. In each case, concentrations of amino acids were greatest when numbers were high. Similarly, comparisons between host species at given times showed that concentrations of amino acids were positively correlated with host selection. In a second study, amino acids of xylem were manipulated by budding scions of a non-preferred host (P. persica) on rootstocks of preferred (P. salinica) and non-preferred (P. persica) hosts. Morphology and phenology of the budded trees were similar to that of the scion species yet the xylem composition of amino acids was primarily dependent on the rootstock. Concentrations of amino acids and the preference of leafhoppers were roughly two-fold greater for scions of the preferred than the non-preferred rootstock. In both studies, amides (glutamine plus asparagine) were the amino acids most highly correlated with host selection. These compounds are the predominant amino acids in xylem fluid, have high nitrogen to carbon ratios, and account for a high percentage of the caloric value in xylem fluid. Many of the less abundant amino acids were positively correlated with host preference, but the correlations were less consistent and correlation coefficients were generally lower.Florida Agricultural Experiment Station Journal Series No. 9672     

The role of sexual isolation during rapid ecological divergence: Evidence for a new dimension of isolation in Rhagoletis pomonella

The pace of divergence and likelihood of speciation often depends on how and when different types of reproductive barriers evolve. Questions remain about how reproductive isolation evolves after initial divergence. We tested for the presence of sexual isolation (reduced mating between populations due to divergent mating preferences and traits) in Rhagoletis pomonella flies, a model system for incipient ecological speciation. We measured the strength of sexual isolation between two very recently diverged (~170 generations) sympatric populations, adapted to different host fruits (hawthorn and apple). We found that flies from both populations were more likely to mate within than between populations. Thus, sexual isolation may play an important role in reducing gene flow allowed by early-acting ecological barriers. We also tested how warmer temperatures predicted under climate change could alter sexual isolation and found that sexual isolation was markedly asymmetric under warmer temperatures – apple males and hawthorn females mated randomly while apple females and hawthorn males mated more within populations than between. Our findings provide a window into the early speciation process and the role of sexual isolation after initial ecological divergence, in addition to examining how environmental conditions could shape the likelihood of further divergence.     

Biological samples taken from Native American Ancestors are human remains under NAGPRA

In the United States, the Native American Graves Protection and Repatriation Act (NAGPRA) provides a specific framework for the disposition of Native American Ancestral remains within its purview. However, samples such as a bone fragment, tooth, or other biological tissue taken from the remains of these Ancestors have been treated by institutions and researchers as independent of the individual from whom they were removed and used in destructive research such as paleogenomic and other archaeometric analyses without consultation, consent, and collaboration from Native American communities; are not cared for in keeping with the current best practices for Indigenous Ancestors; and are not likely to be repatriated to their communities. Here, we demonstrate that any biological samples removed from Ancestors who are covered under NAGPRA must also be handled according to the stipulations defined for “human remains” within the legislation. As such, we are not proposing a change to existing legislation, but rather best practices, specific to the context of the United States and NAGPRA, relating to the use of and care for biological samples taken from Native American Ancestors.     

Genetic mapping of regA mutants of bacteriophage T4D.

SP62, a mutant of bacteriophage T4 shown by Wiberg et al. (1973) to be defective in regulation of T4 protein synthesis, was shown by complementation tests to define a new gene, regA, and by intergenic mapping to lie between genes 43 and 62. The mapping involved crossing SP62 with a quadruple amber mutant defective in genes 42, 43, 62, and 44, selecting all six classes of amber-containing recombinants caused by single crossover events, and then scoring the presence or absence of SP62 in these recombinants. In addition, 15 new, spontaneous regA mutants were isolated, and 13 of these were mapped against each other; a total of eight different mutation sites were thus defined. Most of the new mutants were isolated as pseudorevertants of a leaky amber mutant in gene 62, according to Karam and Bowles (1974), whereas one was identified by virtue of the "white ring" around its plaque, a phenotype possessed by all the regA mutants at high temperature, SP62 was renamed regA1, and the new mutants were named regA2, regA3, etc.     

Ultrastructural events and kinetics of microcyst germination in the myxomycete Didymium iridis

Microcysts of the myxomycete Didymium iridis were induced to excyst by transfer to 5mM potassium phosphate buffer. After 1 h in suspension, 90% of the microcysts had germinated into myxamoebae distinguishable by phase contrast microscopy and staining with Lugol's iodine. Both pH and osmolarity affected the kinetics of excystment. The rate and extent of excystment were decreased by cycloheximide but remained unaffected by actinomycin D, suggesting a requirement for protein synthesis but not RNA synthesis. Initially, the outer wall layers separated from the inner layer, which gradually expanded and loosened. The protoplast rehydrated and reverted to a vegetative morphology. Excysting cells were characterized by nucleolar inclusions, changes in the nuclear envelope and plasma membrane, appearance of ringed cisternal elements and microbodies in the cytoplasm, and formation of a densely fibrous zone adjacent to the site of emergence. Excysting populations have been classified into characteristic stages: mature, initiated, swollen, and pre-emergent microcysts.Florida Agricultural Experiment Station Journal Series No. 2407     

Human monocytes inhibit lymphokine-activated killer cell expansion in vitro

Depleting monocytes from human peripheral blood mononuclear cells (PBMC) enhances the in vitro activation of lymphokine-activated killer (LAK) cells. To determine if monocytes also altered LAK-cell expansion, we evaluated two methods of depleting monocytes from PBMC: nylon wool adherence (NWA) and phenylalanine methyl ester (PME) treatment. Both methods of depleting monocytes enhanced interleukin-2 (IL-2) driven, LAK-cell expansion; LAK expansion, however, was significantly greater after depletion with NWA than after PME. LAK cytotoxicity after NWA and PME depletion was equivalent. The degree of monocyte depletion, determined by evaluating morphology and the number of Leu-M3 (CD14) positive cells, and the proliferation of Leu 19 (CD56), OKT-3 (CD3), Leu2 (CD8), and Leu 3a (CD4) positive cells was also equivalent. Exposure of IL-2 activated cells to PME did not alter their cytotoxic activity. However, sequential treatment of PBMC with NWA, then PME, or with PME and then NWA, resulted in reduced expansion. This reduction in expansion was similar to PBMC treated with PME alone. Exposure of PME-depleted cells to nylon wool or to supernatants obtained from cells adherent to nylon wool further decreased LAK expansion relative to cells treated with NWA alone. We conclude that even at relatively low cell density, human monocytes markedly inhibit LAK-cell expansion in IL-2 driven PBMC cultures. Further, depletion of monocytes by NWA adherence is more effective than by treatment with PME, possibly due to subtle cellular damage induced by this latter treatment. These findings have implication for the in vitro and in vivo generation of LAK-cells by IL-2.